Enzymes and polymerases for the synthesis of rna

ABSTRACT

The invention relates to compositions and methods for the design, evolution, preparation, and/or manufacture of enzymes for use with polynucleotides, primary transcripts and mmRNA molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/682,957, filed Aug. 14, 2012 entitled Synthesis and ChemicalModification of Therapeutic Messenger RNA; U.S. Provisional PatentApplication No. 61/744,806, filed Oct. 3, 2012, entitled Synthesis andChemical Modification of Therapeutic Messenger RNA; U.S. ProvisionalPatent Application No. 61/829,380, filed May 31, 2013, entitledPolymerases; and U.S. Provisional Patent Application No. 61/844,340,filed Jul. 9, 2013, entitled Fusion Enzymes, each of which is hereinincorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing file entitled M032PCT.txt, wascreated on Aug. 13, 2013 and is 100,286 bytes in size. The informationin electronic format of the Sequence Listing is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The invention relates to compositions, methods, processes, for thedesign, evolution, and/or manufacture of enzymes or enzyme variants foruse in the preparation of chemically modified RNA, particularlymessenger RNA. The invention further provides enzymes or enzyme variantsfor the post-transcriptional modification of chemically modified RNA.

BACKGROUND OF THE INVENTION

In the early 1990's Bloom and colleagues successfully rescuedvasopressin-deficient rats by injecting in vitro-transcribed vasopressinmRNA into the hypothalamus (Science 255: 996-998; 1992). However, thelow levels of translation and the immunogenicity of the moleculeshampered the development of mRNA as a therapeutic and efforts have sincefocused on alternative applications that could instead exploit thesepitfalls, i.e. immunization with mRNAs coding for cancer antigens.

Others have investigated the use of mRNA to deliver a polypeptide ofinterest and shown that certain chemical modifications of mRNAmolecules, particularly pseudouridine and 5-methyl-cytosine, havereduced immunostimulatory effect. Notwithstanding these reports whichare limited to a selection of chemical modifications includingpseudouridine and 5-methyl-cytosine, there remains a need in the art fortherapeutic modalities to address the myriad of barriers surrounding theefficacious modulation of intracellular translation and processing ofnucleic acids encoding polypeptides or fragments thereof.

To this end, the inventors have shown that certain modified mRNAsequences have the potential as therapeutics with benefits beyond justevading, avoiding or diminishing the immune response. Such studies aredetailed in published co-pending applications International ApplicationPCT/US2011/046861 filed Aug. 5, 2011 and PCT/US2011/054636 filed Oct. 3,2011, International Application number PCT/US2011/054617 filed Oct. 3,2011, the contents of which are incorporated herein by reference intheir entirety.

The present invention builds upon the aforementioned disclosures andprovides methods and compositions useful in improving the efficiency ofin vitro transcription and translation systems supporting the discoveryand development of chemically modified messenger RNA.

Specifically, the present invention utilizes the PACE (phage-assistedcontinuous directed evolution) method described by Liu for theproduction of novel enzymes and enzyme variants currently used in invitro transcription and post-transcriptional systems (Esvelt et al.(Nature (2011) 472(7344):499-503 and U.S. Publication No. 20110177495).These new enzymes have properties which allow for the production of abroader array of modified messenger RNA molecules, especially thosewhich incorporate unnatural chemical modifications which previouslycould not be incorporated into an mRNA during in vitro transcription.

SUMMARY OF THE INVENTION

Described herein are compositions, methods, processes, for the design,evolution, and/or manufacture of enzymes for use in the preparation ofchemically modified RNA, particularly messenger RNA.

In one aspect of the present invention, provided are chimeric enzymesfor synthesizing capped RNA molecules (e.g., messenger RNA) which mayinclude at least one chemical modification. The chimeric enzyme maycomprise at least one capping enzyme and at least one nucleic acidpolymerase.

The at least one capping enzyme may comprise a Vaccinia capping enzymecomprising a D1 subunit and a D12 subunit or comprises a single subunitcapping enzyme. As a non-limiting example, the D1 subunit may be encodedby the nucleic acid sequence SEQ ID NO: 178 and the D12 subunit may beencoded by the nucleic acid sequence SEQ ID NO: 180. As anothernon-limiting example, the at least one capping enzyme may comprise afirst region of linked nucleosides encoding the D1 subunit having theamino acid sequence SEQ ID NO: 179 and a second region of linkednucleosides encoding the D12 subunit having the amino acid sequence SEQID NO: 181.

In another aspect the chimeric enzyme described herein may comprise anucleic acid polymerase such as, but not limited to, a T7 RNApolymerase, T3 RNA polymerase and SP6 RNA polymerase, a RNA polymerasevariant and a DNA polymerase mutant. The nucleic acid polymerase mayutilize at least one chemical modification into the RNA during in vitrotranscription.

In one aspect, the chimeric enzyme comprises a T7 RNA polymerase. As anon-limiting example, the T7 RNA polymerase may be encoded by thenucleic acid sequence SEQ ID NO: 176 and/or comprises the amino acidsequence SEQ ID NO: 177.

In one aspect, the chimeric enzyme comprises a RNA polymerase variant.As a non-limiting example, the RNA polymerase variant is a T7 RNApolymerase variant. The T7 RNA polymerase variant may be produced byusing continuous evolution such as, but not limited to, phage-assistedcontinuous evolution (PACE). The T7 RNA polymerase variant may be ableto incorporate at least one chemical modification described herein intoRNA during in vitro transcription. The T7 RNA polymerase variant may becharacterized as having an increased transcription efficiency to producemessenger RNA as compared to the wild type T7 RNA polymerase and/or ashaving an increased transcription efficiency through GC-rich regions ascompared to wild type T7 RNA polymerase.

In another aspect, the chimeric enzyme comprises a DNA polymerasemutant. The DNA polymerase mutant may be capable of incorporating atleast one chemical modification described herein. As a non-limitingexample, the DNA polymerase mutant is a DNA polymerase from Thermococcusgorgonarius (Tgo) comprising at least one mutation. The mutation may belocated at an amino acid position such as, but not limited to, 93, 141,143, 403, 409, 485, 657, 658, 659, 663, 664, 669, 671 and 676 of thewild type sequence shown in SEQ ID NO: 32. As a non-limiting example,the mutation may be at position 409 of the wild type sequence and mayinclude the mutation Y409A, Y409G or Y409P. In another non-limitingexample, the mutation may be at position 664 of the wild type sequenceand may include the mutation E664K, E664L, E664Q or E664R. As anon-limiting example, the DNA polymerase mutant comprises the fourmutations, compared to the wild type sequence, of V93Q, D141A, E143A andA485L. As another non-limiting example, the DNA polymerase mutantcomprises thirteen mutations, compared to the wild types sequence, ofV93Q, D141A, E143A, A485L, P657T, E658Q, K659H, Y663H, E664Q, D669A,K671N, T676I and L403P.

In one aspect the chimeric enzyme comprises at least one capping enzymecomprising a Vaccinia capping enzyme catalytic subunit (D1) and aVaccinia capping enzyme stimulation subunit (D12), and the at least onenucleic acid polymerase is T7 RNA polymerase (T7) in one of thefollowing combinations: a first polypeptide comprising D1 linked to T7and a second polypeptide comprising D12; or a first polypeptidecomprising D12 linked to T7 and a second polypeptide comprising D1; or asingle polypeptide comprising D1 linked to D12 linked to T7; or a singlepolypeptide comprising D12 linked to D1 linked to T7; or a singlepolypeptide comprising T7 linked to D1 linked to D12; or a singlepolypeptide comprising T7 linked to D12 linked to D1. The linkages maybe using at least one linker peptide such as, but not limited to,LGGGGSGGGGSGGGGSAAA (SEQ ID NO: 173), LSGGGGSGGGGSGGGGSGGGGSAAA (SEQ IDNO: 174), and EGKSSGSGSESKST (SEQ ID NO: 175), (GGGGS)n, wherein nrefers to any whole integer.

In one aspect, the chimeric enzyme described herein may comprise anaffinity purification tag which optionally may be a His tag.

In another aspect, provided are isolated polynucleotides encoding any ofthe chimeric enzymes described herein.

In one aspect, provided are expression vectors comprising isolatedpolynucleotides encoding the chimeric enzymes described herein. As anon-limiting example, the expression vector may comprise a pEXpressvector or a pET vector.

In another aspect, provided is a host cell comprising an isolatedpolynucleotide encoding the chimeric enzymes described herein or anexpression vector comprising the isolated polynucleotides. As anon-limiting example, the host cell may be a BL21(DE3) cell.

In one aspect, provided are methods of producing chimeric enzymes. As anon-limiting example, the host cell described herein may be culturedunder conditions sufficient to produce a chimeric enzyme. The conditionsmay include culturing a host cell at a temperature below 37 degreesCelsius and/or the solubility of the catalytic subunit of the cappingenzyme is increased compared to expression in the absence of the nucleicacid polymerase. The method may further comprise a purification stepsuch as, but not limited to, an affinity purification, an ion exchangepurification and a size exclusion purification.

In another aspect, provided are methods for synthesizing a capped RNAmolecule comprising at least one chemical modification comprisingcontacting a DNA template with a chimeric enzyme described herein underconditions sufficient to produce a capped RNA molecule.

In one aspect of the present invention, an isolated RNA polymeraseenzyme capable of incorporating chemical modifications into RNA duringin vitro transcription is described. The isolated RNA polymerase may bea T7 RNA polymerase variant, any other RNA polymerase variant describedherein and/or known in the art a DNA polymerase mutant able tosynthesize RNA or any other DNA polymerase mutant able to synthesize RNAdescribed herein and/or known in the art. The RNA polymerase variant maybe capable of incorporating at least one chemical modification selectedfrom the group consisting of pyridin-4-one ribonucleoside,5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-aminopurine,7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine,N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, 2-methoxy-adenine, inosine,1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine,7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine. intoRNA during in vitro transcription. In one embodiment, the RNA is mRNA.

The RNA polymerase enzyme variant, which may be capable of incorporatingchemical modifications, may be produced by continuous evolution such as,but not limited to phage-assisted continuous evolution.

The RNA polymer enzyme variant, may be characterized as having anincreased transcription efficiency through GC-rich regions as comparedto a wild type RNA polymerase, an increased transcription efficiency toproduce mRNA as compared to a wild type RNA polymerase and/or capable ofin vitro transcription at a pH of less than 3 or greater than 7.

In another aspect of the invention, a method of catalyzing the synthesisof RNA is described. A DNA template may be contacted with a RNApolymerase variant capable of incorporating chemical modifications inthe presence of chemically modified nucleoside triphosphates (NTPs). Thechemically modified nucleoside triphosphates (NTPs) may differ fromcytidine, adenosine, guanosine and uridine by a modification to theribofuranosyl ring, by any other modification described herein and/orknown in the art. The NTPs may comprise at least one modificationdescribed herein and/or known in the art. The RNA synthesized may befully modified, at least 50% modified or less than 50% modified.

In yet another aspect of the invention, a method of post-transcriptionalmodification of in vitro or isolated mRNA is described where the invitro or isolated mRNA may be contacted with an enzyme variant producedusing continuous evolution of phage-assisted continuous evolution. Thepost-transcription modification may be methylation which may be modifiedusing the enzyme variant methyl transfersate or pseudouridylation whichmay be modified using the enzyme variant pseudouridine synthetase.

In another aspect of the invention the RNA polymerase variant may be aT7 RNA polymerase variant. The T7 RNA polymerase variant may be producedusing continuous evolution such as, but not limited to phage-assistedcontinuous evolution.

In yet another aspect of the invention the RNA polymerase variant may bea DNA polymerase mutant which can synthesize RNA. The DNA polymerasemutant may be derived from a DNA polymerase from Thermococcusgorgonarius (Tgo). The DNA polymerase mutant may comprise at least onemodification such as, but not limited to, at amino acid position 93,141, 143, 403, 409, 485, 657, 658, 659, 663, 664, 669, 671 and 676 ofthe wild type sequence shown in SEQ ID NO: 32.

In one non-limiting example, the DNA polymerase mutant which is able tosynthesize RNA comprises an amino acid at position 664 of the wild-typesequence, shown in SEQ ID NO: 32, such as, but not limited to, E664K,E664L, E664Q and E664R. In another non-limiting example, the DNApolymerase mutant comprises an amino acid at position 409 of thewild-type sequence, shown in SEQ ID NO: 32, such as, but not limited to,Y409A, Y409G and Y409P.

As a non-limiting example, the DNA polymerase mutant able to synthesizeRNA may comprise the mutations V93Q, D141A, E143A and A485L. In anothernon-limiting example, the DNA polymerase mutant may comprise themutations V93Q, D141A, E143A, A485L, P657T, E658Q, K659H, Y663H, E664Q,D669A, K671N, T676I and L403P of the wild type sequence shown in SEQ IDNO: 32.

In one aspect, a chimeric enzyme may be used for synthesizing andcapping the polynucleotides, primary constructs and/or mmRNA of thepresent invention. The chimeric enzyme may comprise at least one cappingenzyme and at least one polymerase for synthesizing RNA. The cappingenzyme may be Vaccinia capping enzyme (D1 and/or D12) or other enzymesknown in the art. The polymerase may be a DNA polymerase mutated tosynthesize RNA or a RNA polymerase such as, but not limited to, T7 RNApolymerase, T3 RNA polymerase and SP6 RNA polymerase. The chimericenzyme may also be purified before it is used to synthesize and cappolynucleotides, primary constructs and/or mmRNA of the presentinvention.

In another aspect, provided are kit comprising chimeric enzymes andinstructions of use thereof.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews. The drawings are not necessarily to scale, emphasis instead beingplaced upon illustrating the principles of various embodiments of theinvention.

FIG. 1 is a schematic of a primary construct of the present invention.

FIG. 2 represents an overview of the prior art phage-assisted continuousdirected evolution (PACE) system in a single lagoon as taught by Esveltet al (Nature 2011; 472(7344) 499-503).

FIG. 3 is a representative plasmid useful in the IVT reactions taughtherein.

DETAILED DESCRIPTION

It is of great interest in the fields of therapeutics, diagnostics,reagents and for biological assays to be able to deliver a nucleic acid,e.g., a ribonucleic acid (RNA) inside a cell, whether in vitro, in vivo,in situ or ex vivo, such as to cause intracellular translation of thenucleic acid and production of an encoded polypeptide of interest. Ofparticular importance is the delivery and function of a non-integrativepolynucleotide.

Described herein are compositions (including pharmaceuticalcompositions) and methods for the design, preparation, manufactureand/or formulation of polynucleotides encoding one or more polypeptidesof interest. Also provided are systems, processes, devices and kits forthe selection, design and/or utilization of the polynucleotides encodingthe polypeptides of interest described herein.

According to the present invention, these polynucleotides are preferablymodified as to avoid the deficiencies of other polypeptide-encodingmolecules of the art. Hence these polynucleotides are referred to asmodified mRNA or mmRNA. Such modifications, as taught herein, areincorporated into the RNA using enzymes which have been engineered viaphage-assisted continuous evolution (PACE) utilizing n-hybrid methodsand plasmids for mutagenesis (mutagenizing plasmids; MP), accessoryplasmids (AP) and selection phages (SP) which allow selection based onincorporation of modified NTPs.

The use of modified polynucleotides in the fields of antibodies,viruses, veterinary applications and a variety of in vivo settings havebeen explored and these studies are disclosed in for example, co-pendingand co-owned U.S. provisional patent application Ser. No. 61/470,451filed Mar. 31, 2011 teaching in vivo applications of mmRNA; 61/517,784filed on Apr. 26, 2011 teaching engineered nucleic acids for theproduction of antibody polypeptides; 61/519,158 filed May 17, 2011teaching veterinary applications of mmRNA technology; 61/533,537 filedon Sep. 12, 2011 teaching antimicrobial applications of mmRNAtechnology; 61/533,554 filed on Sep. 12, 2011 teaching viralapplications of mmRNA technology, 61/542,533 filed on Oct. 3, 2011teaching various chemical modifications for use in mmRNA technology;61/570,690 filed on Dec. 14, 2011 teaching mobile devices for use inmaking or using mmRNA technology; 61/570,708 filed on Dec. 14, 2011teaching the use of mmRNA in acute care situations; 61/576,651 filed onDec. 16, 2011 teaching terminal modification architecture for mmRNA;61/576,705 filed on Dec. 16, 2011 teaching delivery methods usinglipidoids for mmRNA; 61/578,271 filed on Dec. 21, 2011 teaching methodsto increase the viability of organs or tissues using mmRNA; 61/581,322filed on Dec. 29, 2011 teaching mmRNA encoding cell penetratingpeptides; 61/581,352 filed on Dec. 29, 2011 teaching the incorporationof cytotoxic nucleosides in mmRNA, 61/631,729 filed on Jan. 10, 2012teaching methods of using mmRNA for crossing the blood brain barrier,International Patent Application No PCT/US2012/058519, filed Oct. 3,2012, entitled Modified Nucleosides, Nucleotides, and Nucleic Acids, andUses Thereof, teaching modified nucleosides and nucleotides, andInternational Patent Application No PCT/US2012/069610, filed Dec. 14,2012, entitled Modified Nucleoside, Nucleotide, and Nucleic AcidCompositions, teach formulations of RNA and modified RNA; all of whichare herein incorporated by reference in their entirety.

RNA molecules, such as polynucleotides, primary constructs and mmRNA,which may be synthesized and capped or polypeptides of interest whichmay be encoded by RNA molecules are described in co-pending and co-ownedU.S. Provisional Patent Application No. 61/618,862, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of Biologics, U.S.Provisional Patent Application No. 61/681,645, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Biologics, U.S.Provisional Patent Application No. 61/737,130, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Biologics,International Patent Application No. PCT/US2013/030062, filed Mar. 9,2013, entitled Modified polynucleotides for the Production of Biologicsand Proteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/618,866, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Antibodies, U.S. ProvisionalPatent Application No. 61/681,647, filed Aug. 10, 2012, entitledModified polynucleotides for the Production of Antibodies, U.S.Provisional Patent Application No. 61/737,134, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of Antibodies,International Patent Application No. PCT/US2013/030063, filed Mar. 9,2013, entitled Modified Polynucleotides, U.S. Provisional PatentApplication No. 61/618,868, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Vaccines, U.S. Provisional PatentApplication No. 61/681,648, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Vaccines, U.S. Provisional PatentApplication No. 61/737,135, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Vaccines, U.S. Provisional PatentApplication No. 61/618,870, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Therapeutic Proteins and Peptides,U.S. Provisional Patent Application No. 61/681,649, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of TherapeuticProteins and Peptides, U.S. Provisional Patent Application No.61/737,139, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Therapeutic Proteins and Peptides, U.S. ProvisionalPatent Application No. 61/618,873, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Secreted Proteins, U.S.Provisional Patent Application No. 61/681,650, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of SecretedProteins, U.S. Provisional Patent Application No. 61/737,147, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins, International Patent Application No.PCT/US2013/030064, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Secreted Proteins, U.S. Provisional PatentApplication No. 61/618,878, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Plasma Membrane Proteins, U.S.Provisional Patent Application No. 61/681,654, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins, U.S. Provisional Patent Application No. 61/737,152, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production of PlasmaMembrane Proteins, International Patent Application No.PCT/US2013/030059, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Membrane Proteins, U.S. Provisional PatentApplication No. 61/618,885, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins, U.S. Provisional Patent Application No. 61/681,658, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/737,155, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins, International Patent Application No. PCT/US2013/030066, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/618,896, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrance BoundProteins, U.S. Provisional Patent Application No. 61/668,157, filed Jul.5, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/681,661, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins, U.S. Provisional Patent Application No. 61/737,160, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/618,911, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Nuclear Proteins, U.S. ProvisionalPatent Application No. 61/681,667, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Nuclear Proteins, U.S.Provisional Patent Application No. 61/737,168, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins, International Patent Application No. PCT/US2013/030067, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofNuclear Proteins, U.S. Provisional Patent Application No. 61/618,922,filed Apr. 2, 2012, entitled Modified Polynucleotides for the Productionof Proteins, U.S. Provisional Patent Application No. 61/681,675, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Application No. 61/737,174, filed Dec. 14,2012, entitled Modified Polynucleotides for the Production of Proteins,International Patent Application No PCT/US2013/030060, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of Proteins,U.S. Provisional Patent Application No. 61/618,935, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of ProteinAssociated with Human Disease, U.S. Provisional Patent Application No.61/681,687, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Proteins Associated with Human Disease, U.S. PatentApplication No. 61/737,184, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, International Patent Application No PCT/US2013/030061, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/618,945, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/681,696, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/737,191, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/618,953, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/681,704, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/737,203, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, International Patent ApplicationNo PCT/US2013/031821, filed Mar. 15, 2013, entitled In Vivo Productionof Proteins, U.S. Provisional Patent Application No. 61/681,720, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofCosmetic Proteins and Peptides, U.S. Provisional Patent Application No.61/737,213, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Cosmetic Proteins and Peptides, International PatentApplication No PCT/US2013/030068, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Cosmetic Proteins and Peptides,U.S. Provisional Patent Application No. 61/681,742, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Oncology-RelatedProteins and Peptides, International Patent Application NoPCT/US2013/030070, filed Mar. 9, 2012, entitled Modified Polynucleotidesfor the Production of Oncology-Related Proteins and Peptides, thecontents each of which are herein incorporated by reference in theirentireties.

For each of these inventions, the design of enzymes having superiorproperties such as permissivity regarding incorporation of a variety ofNTPs will provide further advances in therapeutics as well asdiagnostics.

Provided herein, are polynucleotides, primary constructs and/or mmRNAencoding polypeptides of interest which have been designed to improveone or more of the stability and/or clearance in tissues, receptoruptake and/or kinetics, cellular access by the compositions, engagementwith translational machinery, mRNA half-life, translation efficiency,immune evasion, protein production capacity, secretion efficiency (whenapplicable), accessibility to circulation, protein half-life and/ormodulation of a cell's status, function and/or activity. Theseproperties arise, in part, from the incorporation of modifications whichhave not before been possible using standard or wild type enzymes. mmRNAcreated in this manner are described below.

I. COMPOSITIONS OF THE INVENTION (mmRNA)

The present invention provides nucleic acid molecules, specificallypolynucleotides, primary constructs and/or mmRNA which encode one ormore polypeptides of interest. The nucleic acid molecules orpolynucleotides or any portion or region thereof may be synthesized ormodified using the novel enzymes or enzyme variants of the invention.

The term “nucleic acid,” in its broadest sense, includes any compoundand/or substance that comprise a polymer of nucleotides. These polymersare often referred to as polynucleotides. Exemplary nucleic acids orpolynucleotides of the invention include, but are not limited to,ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleicacids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs),locked nucleic acids (LNAs, including LNA having a β-D-riboconfiguration, α-LNA having an α-L-ribo configuration (a diastereomer ofLNA), 2′-amino-LNA having a 2′-amino functionalization, and2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof.

In preferred embodiments, the nucleic acid molecule is a messenger RNA(mRNA). As used herein, the term “messenger RNA” (mRNA) refers to anypolynucleotide which encodes a polypeptide of interest and which iscapable of being translated to produce the encoded polypeptide ofinterest in vitro, in vivo, in situ or ex vivo.

Traditionally, the basic components of an mRNA molecule include at leasta coding region, a 5′UTR, a 3′UTR, a 5′ cap and a poly-A tail. Buildingon this wild type modular structure, the present invention expands thescope of functionality of traditional mRNA molecules by providingpolynucleotides or primary RNA constructs which maintain a modularorganization, but which comprise one or more structural and/or chemicalmodifications or alterations which impart useful properties to thepolynucleotide including, in some embodiments, the lack of a substantialinduction of the innate immune response of a cell into which thepolynucleotide is introduced. As such, modified mRNA molecules of thepresent invention are termed “mmRNA.” As used herein, a “structural”feature or modification is one in which two or more linked nucleotidesare inserted, deleted, duplicated, inverted or randomized in apolynucleotide, primary construct or mmRNA without significant chemicalmodification to the nucleotides themselves. Because chemical bonds willnecessarily be broken and reformed to effect a structural modification,structural modifications are of a chemical nature and hence are chemicalmodifications. However, structural modifications will result in adifferent sequence of nucleotides. For example, the polynucleotide“ATCG” may be chemically modified to “AT-5meC-G”. The samepolynucleotide may be structurally modified from “ATCG” to “ATCCCG”.Here, the dinucleotide “CC” has been inserted, resulting in a structuralmodification to the polynucleotide.

mmRNA Architecture

The mmRNA of the present invention are distinguished from wild type mRNAin their functional and/or structural design features which serve to, asevidenced herein, overcome existing problems of effective polypeptideproduction using nucleic acid-based therapeutics.

FIG. 1 shows a representative polynucleotide primary construct 100 ofthe present invention. As used herein, the term “primary construct” or“primary mRNA construct” refers to a polynucleotide transcript whichencodes one or more polypeptides of interest and which retainssufficient structural and/or chemical features to allow the polypeptideof interest encoded therein to be translated. Primary constructs may bepolynucleotides of the invention. When structurally or chemicallymodified, the primary construct may be referred to as an mmRNA.

Returning to FIG. 1, the primary construct 100 here contains a firstregion of linked nucleotides 102 that is flanked by a first flankingregion 104 and a second flaking region 106. As used herein, the “firstregion” may be referred to as a “coding region” or “region encoding” orsimply the “first region.” This first region may include, but is notlimited to, the encoded polypeptide of interest. The polypeptide ofinterest may comprise at its 5′ terminus one or more signal sequencesencoded by a signal sequence region 103. The flanking region 104 maycomprise a region of linked nucleotides comprising one or more completeor incomplete 5′ UTRs sequences. The flanking region 104 may alsocomprise a 5′ terminal cap 108. The second flanking region 106 maycomprise a region of linked nucleotides comprising one or more completeor incomplete 3′ UTRs. The flanking region 106 may also comprise a 3′tailing sequence 110.

Bridging the 5′ terminus of the first region 102 and the first flankingregion 104 is a first operational region 105. Traditionally thisoperational region comprises a Start codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a Start codon.

Bridging the 3′ terminus of the first region 102 and the second flankingregion 106 is a second operational region 107. Traditionally thisoperational region comprises a Stop codon. The operational region mayalternatively comprise any translation initiation sequence or signalincluding a Stop codon. According to the present invention, multipleserial stop codons may also be used.

Generally, the shortest length of the first region of the primaryconstruct of the present invention can be the length of a nucleic acidsequence that is sufficient to encode for a dipeptide, a tripeptide, atetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, anoctapeptide, a nonapeptide, or a decapeptide. In another embodiment, thelength may be sufficient to encode a peptide of 2-30 amino acids, e.g.5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may besufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17,20, 25 or 30 amino acids, or a peptide that is no longer than 40 aminoacids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10amino acids. Examples of dipeptides that the polynucleotide sequencescan encode or include, but are not limited to, carnosine and anserine.

Generally, the length of the first region encoding the polypeptide ofinterest of the present invention is greater than about 30 nucleotidesin length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600,1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000,7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000,70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). Asused herein, the “first region” may be referred to as a “coding region”or “region encoding” or simply the “first region.”

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes from about 30 to about 100,000 nucleotides (e.g., from 30 to50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000,from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000,from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000,from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000,from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000,from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to70,000, and from 2,000 to 100,000).

According to the present invention, the first and second flankingregions may range independently from 15-1,000 nucleotides in length(e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,and 1,000 nucleotides).

According to the present invention, the tailing sequence may range fromabsent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120,140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Wherethe tailing region is a polyA tail, the length may be determined inunits of or as a function of polyA Binding Protein binding. In thisembodiment, the polyA tail is long enough to bind at least 4 monomers ofPolyA Binding Protein. PolyA Binding Protein monomers bind to stretchesof approximately 38 nucleotides. As such, it has been observed thatpolyA tails of about 80 nucleotides and 160 nucleotides are functional.

According to the present invention, the capping region may comprise asingle cap or a series of nucleotides forming the cap. In thisembodiment the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7,1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In someembodiments, the cap is absent.

According to the present invention, the first and second operationalregions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or30 or fewer nucleotides in length and may comprise, in addition to aStart and/or Stop codon, one or more signal and/or restrictionsequences.

Cyclic mmRNA

According to the present invention, a primary construct or mmRNA may becyclized, or concatemerized, to generate a translation competentmolecule to assist interactions between poly-A binding proteins and5′-end binding proteins. The mechanism of cyclization orconcatemerization may occur through at least 3 different routes: 1)chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed5′-/3′-linkage may be intramolecular or intermolecular.

In the first route, the 5′-end and the 3′-end of the nucleic acidcontain chemically reactive groups that, when close together, form a newcovalent linkage between the 5′-end and the 3′-end of the molecule. The5′-end may contain an NHS-ester reactive group and the 3′-end maycontain a 3′-amino-terminated nucleotide such that in an organic solventthe 3′-amino-terminated nucleotide on the 3′-end of a synthetic mRNAmolecule will undergo a nucleophilic attack on the 5′-NHS-ester moietyforming a new 5′-/3′-amide bond.

In the second route, T4 RNA ligase or a ligase variant evolved usingPACE may be used to enzymatically link a 5′-phosphorylated nucleic acidmolecule to the 3′-hydroxyl group of a nucleic acid forming a newphosphorodiester linkage. In an example reaction, 1 μg of a nucleic acidmolecule is incubated at 37° C. for 1 hour with 1-10 units of T4 RNAligase (New England Biolabs, Ipswich, Mass.) according to themanufacturer's protocol. The ligation reaction may occur in the presenceof a split oligonucleotide capable of base-pairing with both the 5′- and3′-region in juxtaposition to assist the enzymatic ligation reaction.

In the third route, either the 5′- or 3′-end of the cDNA templateencodes a ligase ribozyme sequence such that during in vitrotranscription, the resultant nucleic acid molecule can contain an activeribozyme sequence capable of ligating the 5′-end of a nucleic acidmolecule to the 3′-end of a nucleic acid molecule. The ligase ribozymemay be derived from the Group I Intron, Group I Intron, Hepatitis DeltaVirus, Hairpin ribozyme or may be selected by SELEX (systematicevolution of ligands by exponential enrichment). The ribozyme ligasereaction may take 1 to 24 hours at temperatures between 0 and 37° C.

mmRNA Multimers

According to the present invention, multiple distinct polynucleotides,primary constructs or mmRNA may be linked together through the 3′-endusing nucleotides which are modified at the 3′-terminus. Chemicalconjugation may be used to control the stoichiometry of delivery intocells. For example, the glyoxylate cycle enzymes, isocitrate lyase andmalate synthase, may be supplied into HepG2 cells at a 1:1 ratio toalter cellular fatty acid metabolism. This ratio may be controlled bychemically linking polynucleotides, primary constructs or mmRNA using a3′-azido terminated nucleotide on one polynucleotide, primary constructor mmRNA species and a C5-ethynyl or alkynyl-containing nucleotide onthe opposite polynucleotide, primary construct or mmRNA species. Themodified nucleotide is added post-transcriptionally using terminaltransferase (New England Biolabs, Ipswich, Mass.) according to themanufacturer's protocol. After the addition of the 3′-modifiednucleotide, the two polynucleotide, primary construct or mmRNA speciesmay be combined in an aqueous solution, in the presence or absence ofcopper, to form a new covalent linkage via a click chemistry mechanismas described in the literature.

In another example, more than two polynucleotides may be linked togetherusing a functionalized linker molecule. For example, a functionalizedsaccharide molecule may be chemically modified to contain multiplechemical reactive groups (SH—, NH₂—, N₃, etc . . . ) to react with thecognate moiety on a 3′-functionalized mRNA molecule (i.e., a3′-maleimide ester, 3′-NHS-ester, alkynyl). The number of reactivegroups on the modified saccharide can be controlled in a stoichiometricfashion to directly control the stoichiometric ratio of conjugatedpolynucleotide, primary construct or mmRNA.

mmRNA Conjugates and Combinations

In order to further enhance protein production, primary constructs ormmRNA of the present invention can be designed to be conjugated to otherpolynucleotides, dyes, intercalating agents (e.g. acridines),cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K),MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeledmarkers, enzymes, haptens (e.g. biotin), transport/absorptionfacilitators (e.g., aspirin, vitamin E, folic acid), syntheticribonucleases, proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell, hormones and hormone receptors,non-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, or a drug.

Conjugation may result in increased stability and/or half life and maybe particularly useful in targeting the polynucleotides, primaryconstructs or mmRNA to specific sites in the cell, tissue or organism.

According to the present invention, the mmRNA or primary constructs maybe administered with, or further encode one or more of RNAi agents,siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes,catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamersor vectors, and the like.

Bifunctional mmRNA

In one embodiment of the invention are bifunctional polynucleotides(e.g., bifunctional primary constructs or bifunctional mmRNA). As thename implies, bifunctional polynucleotides are those having or capableof at least two functions. These molecules may also by convention bereferred to as multi-functional.

The multiple functionalities of bifunctional polynucleotides may beencoded by the RNA (the function may not manifest until the encodedproduct is translated) or may be a property of the polynucleotideitself. It may be structural or chemical. Bifunctional modifiedpolynucleotides may comprise a function that is covalently orelectrostatically associated with the polynucleotides. Further, the twofunctions may be provided in the context of a complex of a mmRNA andanother molecule.

Bifunctional polynucleotides may encode peptides which areanti-proliferative. These peptides may be linear, cyclic, constrained orrandom coil. They may function as aptamers, signaling molecules, ligandsor mimics or mimetics thereof. Anti-proliferative peptides may, astranslated, be from 3 to 50 amino acids in length. They may be 5-40,10-30, or approximately 15 amino acids long. They may be single chain,multichain or branched and may form complexes, aggregates or anymulti-unit structure once translated.

Noncoding Polynucleotides and Primary Constructs

As described herein, provided are polynucleotides and primary constructshaving sequences that are partially or substantially not translatable,e.g., having a noncoding region. Such noncoding region may be the “firstregion” of the primary construct. Alternatively, the noncoding regionmay be a region other than the first region. Such molecules aregenerally not translated, but can exert an effect on protein productionby one or more of binding to and sequestering one or more translationalmachinery components such as a ribosomal protein or a transfer RNA(tRNA), thereby effectively reducing protein expression in the cell ormodulating one or more pathways or cascades in a cell which in turnalters protein levels. The polynucleotide or primary construct maycontain or encode one or more long noncoding RNA (lncRNA, or lincRNA) orportion thereof, a small nucleolar RNA (sno-RNA), micro RNA (miRNA),small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).

Polypeptides of Interest

According to the present invention, the primary construct is designed toencode one or more polypeptides of interest or fragments thereof. Apolypeptide of interest may include, but is not limited to, wholepolypeptides, a plurality of polypeptides or fragments of polypeptides,which independently may be encoded by one or more nucleic acids, aplurality of nucleic acids, fragments of nucleic acids or variants ofany of the aforementioned. As used herein, the term “polypeptides ofinterest” refer to any polypeptide which is selected to be encoded inthe primary construct of the present invention.

In the development of novel enzymes and enzyme variants, the polypeptideof interest may encode a signaling molecule or binding partnersufficient to, when expressed during PACE, trigger the expression of theaccessory plasmid (AP) allowing for positive selection of enzymessatisfying the selection criteria. For example, when evolving novelpolymerases which are permissive to chemically modified NTPs, thepolypeptide of interest (encoded by an RNA or mRNA containing novelchemically modified nucleotides) may be one which triggers theexpression of the accessory plasmid thereby selecting for the polymerasewhich first incorporated the modifications which could not beincorporated by native or wild type polymerases. Such n-hybrid systemsare well known in the art and can be selected based on the design of theselection phage which incorporates the library molecules of potentialenzyme variants being screened.

As used herein, “polypeptide” means a polymer of amino acid residues(natural or unnatural) linked together most often by peptide bonds. Theterm, as used herein, refers to proteins, polypeptides, and peptides ofany size, structure, or function. In some instances the polypeptideencoded is smaller than about 50 amino acids and the polypeptide is thentermed a peptide. If the polypeptide is a peptide, it will be at leastabout 2, 3, 4, or at least 5 amino acid residues long. Thus,polypeptides include gene products, naturally occurring polypeptides,synthetic polypeptides, homologs, orthologs, paralogs, fragments andother equivalents, variants, and analogs of the foregoing. A polypeptidemay be a single molecule or may be a multi-molecular complex such as adimer, trimer or tetramer. They may also comprise single chain ormultichain polypeptides such as antibodies or insulin and may beassociated or linked. Most commonly disulfide linkages are found inmultichain polypeptides. The term polypeptide may also apply to aminoacid polymers in which one or more amino acid residues are an artificialchemical analogue of a corresponding naturally occurring amino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants willpossess at least about 50% identity (homology) to a native or referencesequence, and preferably, they will be at least about 80%, morepreferably at least about 90% identical (homologous) to a native orreference sequence. The “enzyme variants” of the present invention arealso polypeptide variants but with additional catalytic activity.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains one or more amino acids whichwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, e.g., phenylalanine may act as an inactivatingsubstitution for tyrosine; or alanine may act as an inactivatingsubstitution for serine.

“Homology” as it applies to amino acid sequences is defined as thepercentage of residues in the candidate amino acid sequence that areidentical with the residues in the amino acid sequence of a secondsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well known in the art. It is understoodthat homology depends on a calculation of percent identity but maydiffer in value due to gaps and penalties introduced in the calculation.

By “homologs” as it applies to polypeptide sequences means thecorresponding sequence of other species having substantial identity to asecond sequence of a second species.

“Analogs” is meant to include polypeptide variants which differ by oneor more amino acid alterations, e.g., substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present invention contemplates several types of compositions whichare polypeptide based including variants and derivatives. These includesubstitutional, insertional, deletion and covalent variants andderivatives. The term “derivative” is used synonymously with the term“variant” but generally refers to a molecule that has been modifiedand/or changed in any way relative to a reference molecule or startingmolecule.

As such, mmRNA encoding polypeptides containing substitutions,insertions and/or additions, deletions and covalent modifications withrespect to reference sequences, in particular the polypeptide sequencesdisclosed herein, are included within the scope of this invention. Forexample, sequence tags or amino acids, such as one or more lysines, canbe added to the peptide sequences of the invention (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidepurification or localization. Lysines can be used to increase peptidesolubility or to allow for biotinylation. Alternatively, amino acidresidues located at the carboxy and amino terminal regions of the aminoacid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalor N-terminal residues) may alternatively be deleted depending on theuse of the sequence, as for example, expression of the sequence as partof a larger sequence which is soluble, or linked to a solid support.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. The substitutions may be single, where only one amino acid inthe molecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Insertional variants” when referring to polypeptides are those with oneor more amino acids inserted immediately adjacent to an amino acid at aparticular position in a native or starting sequence. “Immediatelyadjacent” to an amino acid means connected to either the alpha-carboxyor alpha-amino functional group of the amino acid.

“Deletional variants” when referring to polypeptides are those with oneor more amino acids in the native or starting amino acid sequenceremoved. Ordinarily, deletional variants will have one or more aminoacids deleted in a particular region of the molecule.

“Covalent derivatives” when referring to polypeptides includemodifications of a native or starting protein with an organicproteinaceous or non-proteinaceous derivatizing agent, and/orpost-translational modifications. Covalent modifications aretraditionally introduced by reacting targeted amino acid residues of theprotein with an organic derivatizing agent that is capable of reactingwith selected side-chains or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. The resultant covalent derivatives are useful inprograms directed at identifying residues important for biologicalactivity, for immunoassays, or for the preparation of anti-proteinantibodies for immunoaffinity purification of the recombinantglycoprotein. Such modifications are within the ordinary skill in theart and are performed without undue experimentation.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues may be present in the polypeptides produced in accordancewith the present invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the alpha-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)).

“Features” when referring to polypeptides are defined as distinct aminoacid sequence-based components of a molecule. Features of thepolypeptides encoded by the mmRNA of the present invention includesurface manifestations, local conformational shape, folds, loops,half-loops, domains, half-domains, sites, termini or any combinationthereof.

As used herein when referring to polypeptides the term “surfacemanifestation” refers to a polypeptide based component of a proteinappearing on an outermost surface.

As used herein when referring to polypeptides the term “localconformational shape” means a polypeptide based structural manifestationof a protein which is located within a definable space of the protein.

As used herein when referring to polypeptides the term “fold” refers tothe resultant conformation of an amino acid sequence upon energyminimization. A fold may occur at the secondary or tertiary level of thefolding process. Examples of secondary level folds include beta sheetsand alpha helices. Examples of tertiary folds include domains andregions formed due to aggregation or separation of energetic forces.Regions formed in this way include hydrophobic and hydrophilic pockets,and the like.

As used herein the term “turn” as it relates to protein conformationmeans a bend which alters the direction of the backbone of a peptide orpolypeptide and may involve one, two, three or more amino acid residues.

As used herein when referring to polypeptides the term “loop” refers toa structural feature of a polypeptide which may serve to reverse thedirection of the backbone of a peptide or polypeptide. Where the loop isfound in a polypeptide and only alters the direction of the backbone, itmay comprise four or more amino acid residues. Oliva et al. haveidentified at least 5 classes of protein loops (J. Mol Biol 266 (4):814-830; 1997). Loops may be open or closed. Closed loops or “cyclic”loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acidsbetween the bridging moieties. Such bridging moieties may comprise acysteine-cysteine bridge (Cys-Cys) typical in polypeptides havingdisulfide bridges or alternatively bridging moieties may be non-proteinbased such as the dibromozylyl agents used herein.

As used herein when referring to polypeptides the term “half-loop”refers to a portion of an identified loop having at least half thenumber of amino acid resides as the loop from which it is derived. It isunderstood that loops may not always contain an even number of aminoacid residues. Therefore, in those cases where a loop contains or isidentified to comprise an odd number of amino acids, a half-loop of theodd-numbered loop will comprise the whole number portion or next wholenumber portion of the loop (number of amino acids of the loop/2+/−0.5amino acids). For example, a loop identified as a 7 amino acid loopcould produce half-loops of 3 amino acids or 4 amino acids(7/2=3.5+/−0.5 being 3 or 4).

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the term “half-domain”means a portion of an identified domain having at least half the numberof amino acid resides as the domain from which it is derived. It isunderstood that domains may not always contain an even number of aminoacid residues. Therefore, in those cases where a domain contains or isidentified to comprise an odd number of amino acids, a half-domain ofthe odd-numbered domain will comprise the whole number portion or nextwhole number portion of the domain (number of amino acids of thedomain/2+/−0.5 amino acids). For example, a domain identified as a 7amino acid domain could produce half-domains of 3 amino acids or 4 aminoacids (7/2=3.5+/−0.5 being 3 or 4). It is also understood thatsub-domains may be identified within domains or half-domains, thesesubdomains possessing less than all of the structural or functionalproperties identified in the domains or half domains from which theywere derived. It is also understood that the amino acids that compriseany of the domain types herein need not be contiguous along the backboneof the polypeptide (i.e., nonadjacent amino acids may fold structurallyto produce a domain, half-domain or subdomain).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” A site represents aposition within a peptide or polypeptide that may be modified,manipulated, altered, derivatized or varied within the polypeptide basedmolecules of the present invention.

As used herein the terms “termini” or “terminus” when referring topolypeptides refers to an extremity of a peptide or polypeptide. Suchextremity is not limited only to the first or final site of the peptideor polypeptide but may include additional amino acids in the terminalregions. The polypeptide based molecules of the present invention may becharacterized as having both an N-terminus (terminated by an amino acidwith a free amino group (NH2)) and a C-terminus (terminated by an aminoacid with a free carboxyl group (COOH)). Proteins of the invention arein some cases made up of multiple polypeptide chains brought together bydisulfide bonds or by non-covalent forces (multimers, oligomers). Thesesorts of proteins will have multiple N- and C-termini. Alternatively,the termini of the polypeptides may be modified such that they begin orend, as the case may be, with a non-polypeptide based moiety such as anorganic conjugate.

Once any of the features have been identified or defined as a desiredcomponent of a polypeptide to be encoded by the primary construct ormmRNA of the invention, any of several manipulations and/ormodifications of these features may be performed by moving, swapping,inverting, deleting, randomizing or duplicating. Furthermore, it isunderstood that manipulation of features may result in the same outcomeas a modification to the molecules of the invention. For example, amanipulation which involved deleting a domain would result in thealteration of the length of a molecule just as modification of a nucleicacid to encode less than a full length molecule would.

Modifications and manipulations can be accomplished by methods known inthe art such as, but not limited to, site directed mutagenesis. Theresulting modified molecules may then be tested for activity using invitro or in vivo assays such as those described herein or any othersuitable screening assay known in the art.

According to the present invention, the polypeptides may comprise aconsensus sequence which is discovered through rounds ofexperimentation. As used herein a “consensus” sequence is a singlesequence which represents a collective population of sequences allowingfor variability at one or more sites.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest of this invention. Forexample, provided herein is any protein fragment (meaning a polypeptidesequence at least one amino acid residue shorter than a referencepolypeptide sequence but otherwise identical) of a reference protein 10,20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids inlength. In another example, any protein that includes a stretch of about20, about 30, about 40, about 50, or about 100 amino acids which areabout 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, or about 100% identical to any of the sequences described hereincan be utilized in accordance with the invention. In certainembodiments, a polypeptide to be utilized in accordance with theinvention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations asshown in any of the sequences provided or referenced herein.

Encoded Polypeptides

The primary constructs or mmRNA of the present invention may be designedto encode polypeptides of interest selected from any of several targetcategories including, but not limited to, biologics, antibodies,vaccines, therapeutic proteins or peptides, cell penetrating peptides,n-hybrid system proteins or peptides and/or binding partner or signalingmolecules useful in PACE systems, secreted proteins, plasma membraneproteins, cytoplasmic or cytoskeletal proteins, intracellular membranebound proteins, nuclear proteins, proteins associated with humandisease, targeting moieties or those proteins encoded by the humangenome for which no therapeutic indication has been identified but whichnonetheless have utility in areas of research and discovery.

In one embodiment primary constructs or mmRNA may encode variantpolypeptides which have a certain identity with a reference polypeptidesequence. As used herein, a “reference polypeptide sequence” refers to astarting polypeptide sequence. Reference sequences may be wild typesequences or any sequence to which reference is made in the design ofanother sequence.

The term “identity” as known in the art, refers to a relationshipbetween the sequences of two or more peptides, as determined bycomparing the sequences. In the art, identity also means the degree ofsequence relatedness between peptides, as determined by the number ofmatches between strings of two or more amino acid residues. Identitymeasures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated peptides can be readily calculated by known methods. Suchmethods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

In some embodiments, the polypeptide variant may have the same or asimilar activity as the reference polypeptide. Alternatively, thevariant may have an altered activity (e.g., increased or decreased)relative to a reference polypeptide. Generally, variants of a particularpolynucleotide or polypeptide of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity tothat particular reference polynucleotide or polypeptide as determined bysequence alignment programs and parameters described herein and known tothose skilled in the art. Such tools for alignment include those of theBLAST suite (Stephen F. Altschul, Thomas L. Madden, Alejandro A.Schïffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman(1997), “Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs”, Nucleic Acids Res. 25:3389-3402.) Other toolsare described herein, specifically in the definition of “Identity.”

Default parameters in the BLAST algorithm include, for example, anexpect threshold of 10, Word size of 28, Match/Mismatch Scores 1, −2,Gap costs Linear. Any filter can be applied as well as a selection forspecies specific repeats, e.g., Homo sapiens.

Biologics

The polynucleotides, primary constructs or mmRNA disclosed herein, mayencode one or more biologics. As used herein, a “biologic” is apolypeptide-based molecule produced by the methods provided herein andwhich may be used to treat, cure, mitigate, prevent, or diagnose aserious or life-threatening disease or medical condition. Biologics,according to the present invention include, but are not limited to,allergenic extracts (e.g. for allergy shots and tests), bloodcomponents, gene therapy products, human tissue or cellular productsused in transplantation, vaccines, monoclonal antibodies, cytokines,growth factors, enzymes, thrombolytics, and immunomodulators, amongothers.

According to the present invention, one or more biologics currentlybeing marketed or in development may be encoded by the polynucleotides,primary constructs or mmRNA of the present invention. While not wishingto be bound by theory, it is believed that incorporation of the encodingpolynucleotides of a known biologic into the primary constructs or mmRNAof the invention will result in improved therapeutic efficacy due atleast in part to the specificity, purity and/or selectivity of theconstruct designs.

Antibodies

The primary constructs or mmRNA disclosed herein, may encode one or moreantibodies or fragments thereof. The term “antibody” includes monoclonalantibodies (including full length antibodies which have animmunoglobulin Fc region), antibody compositions with polyepitopicspecificity, multispecific antibodies (e.g., bispecific antibodies,diabodies, and single-chain molecules), as well as antibody fragments.The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein. As used herein, the term “monoclonal antibody” refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity. Chimericantibodies of interest herein include, but are not limited to,“primatized” antibodies comprising variable domain antigen-bindingsequences derived from a non-human primate (e.g., Old World Monkey, Apeetc.) and human constant region sequences.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies; nanobodies; single-chainantibody molecules and multispecific antibodies formed from antibodyfragments.

Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM,may be encoded by the mmRNA of the invention, including the heavy chainsdesignated alpha, delta, epsilon, gamma and mu, respectively. Alsoincluded are polynucleotide sequences encoding the subclasses, gamma andmu. Hence any of the subclasses of antibodies may be encoded in part orin whole and include the following subclasses: IgG1, IgG2, IgG3, IgG4,IgA1 and IgA2.

According to the present invention, one or more antibodies or fragmentscurrently being marketed or in development may be encoded by thepolynucleotides, primary constructs or mmRNA of the present invention.While not wishing to be bound by theory, it is believed thatincorporation into the primary constructs of the invention will resultin improved therapeutic efficacy due at least in part to thespecificity, purity and selectivity of the mmRNA designs.

Antibodies encoded in the polynucleotides, primary constructs or mmRNAof the invention may be utilized to treat conditions or diseases in manytherapeutic areas such as, but not limited to, blood, cardiovascular,CNS, poisoning (including antivenoms), dermatology, endocrinology,gastrointestinal, medical imaging, musculoskeletal, oncology,immunology, respiratory, sensory and anti-infective.

In one embodiment, primary constructs or mmRNA disclosed herein mayencode monoclonal antibodies and/or variants thereof. Variants ofantibodies may also include, but are not limited to, substitutionalvariants, conservative amino acid substitution, insertional variants,deletional variants and/or covalent derivatives. In one embodiment, theprimary construct and/or mmRNA disclosed herein may encode animmunoglobulin Fc region. In another embodiment, the primary constructsand/or mmRNA may encode a variant immunoglobulin Fc region. As anon-limiting example, the primary constructs and/or mmRNA may encode anantibody having a variant immunoglobulin Fc region as described in U.S.Pat. No. 8,217,147 herein incorporated by reference in its entirety.

Vaccines

The primary constructs or mmRNA disclosed herein, may encode one or morevaccines. As used herein, a “vaccine” is a biological preparation thatimproves immunity to a particular disease or infectious agent. Accordingto the present invention, one or more vaccines currently being marketedor in development may be encoded by the polynucleotides, primaryconstructs or mmRNA of the present invention. While not wishing to bebound by theory, it is believed that incorporation into the primaryconstructs or mmRNA of the invention will result in improved therapeuticefficacy due at least in part to the specificity, purity and selectivityof the construct designs.

Vaccines encoded in the polynucleotides, primary constructs or mmRNA ofthe invention may be utilized to treat conditions or diseases in manytherapeutic areas such as, but not limited to, cardiovascular, CNS,dermatology, endocrinology, oncology, immunology, respiratory, andanti-infective.

Therapeutic Proteins or Peptides

The primary constructs or mmRNA disclosed herein, may encode one or morevalidated or “in testing” therapeutic proteins or peptides.

According to the present invention, one or more therapeutic proteins orpeptides currently being marketed or in development may be encoded bythe polynucleotides, primary constructs or mmRNA of the presentinvention. While not wishing to be bound by theory, it is believed thatincorporation into the primary constructs or mmRNA of the invention willresult in improved therapeutic efficacy due at least in part to thespecificity, purity and selectivity of the construct designs.

Therapeutic proteins and peptides encoded in the polynucleotides,primary constructs or mmRNA of the invention may be utilized to treatconditions or diseases in many therapeutic areas such as, but notlimited to, blood, cardiovascular, CNS, poisoning (includingantivenoms), dermatology, endocrinology, genetic, genitourinary,gastrointestinal, musculoskeletal, oncology, and immunology,respiratory, sensory and anti-infective.

Cell-Penetrating Polypeptides

The primary constructs or mmRNA disclosed herein, may encode one or morecell-penetrating polypeptides. As used herein, “cell-penetratingpolypeptide” or CPP refers to a polypeptide which may facilitate thecellular uptake of molecules. A cell-penetrating polypeptide of thepresent invention may contain one or more detectable labels. Thepolypeptides may be partially labeled or completely labeled throughout.The polynucleotide, primary construct or mmRNA may encode the detectablelabel completely, partially or not at all. The cell-penetrating peptidemay also include a signal sequence. As used herein, a “signal sequence”refers to a sequence of amino acid residues bound at the amino terminusof a nascent protein during protein translation. The signal sequence maybe used to signal the secretion of the cell-penetrating polypeptide.

In one embodiment, the polynucleotides, primary constructs or mmRNA mayalso encode a fusion protein. The fusion protein may be created byoperably linking a charged protein to a therapeutic protein. As usedherein, “operably linked” refers to the therapeutic protein and thecharged protein being connected in such a way to permit the expressionof the complex when introduced into the cell. As used herein, “chargedprotein” refers to a protein that carries a positive, negative oroverall neutral electrical charge. Preferably, the therapeutic proteinmay be covalently linked to the charged protein in the formation of thefusion protein. The ratio of surface charge to total or surface aminoacids may be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or0.9.

The cell-penetrating polypeptide encoded by the polynucleotides, primaryconstructs or mmRNA may form a complex after being translated. Thecomplex may comprise a charged protein linked, e.g. covalently linked,to the cell-penetrating polypeptide. “Therapeutic protein” refers to aprotein that, when administered to a cell has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but is not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the polynucleotide, primary construct or mmRNA may beintroduced. The cell-penetrating polypeptide may also be capable ofpenetrating the first cell.

In a further embodiment, the cell-penetrating polypeptide is capable ofpenetrating a second cell. The second cell may be from the same area asthe first cell, or it may be from a different area. The area mayinclude, but is not limited to, tissues and organs. The second cell mayalso be proximal or distal to the first cell.

In one embodiment, the polynucleotides, primary constructs or mmRNA mayencode a cell-penetrating polypeptide which may comprise aprotein-binding partner. The protein binding partner may include, but isnot limited to, an antibody, a supercharged antibody or a functionalfragment. The polynucleotides, primary constructs or mmRNA may beintroduced into the cell where a cell-penetrating polypeptide comprisingthe protein-binding partner is introduced.

Secreted Proteins

Human and other eukaryotic cells are subdivided by membranes into manyfunctionally distinct compartments. Each membrane-bounded compartment,or organelle, contains different proteins essential for the function ofthe organelle. The cell uses “sorting signals,” which are amino acidmotifs located within the protein, to target proteins to particularcellular organelles.

One type of sorting signal, called a signal sequence, a signal peptide,or a leader sequence, directs a class of proteins to an organelle calledthe endoplasmic reticulum (ER).

Proteins targeted to the ER by a signal sequence can be released intothe extracellular space as a secreted protein. Similarly, proteinsresiding on the cell membrane can also be secreted into theextracellular space by proteolytic cleavage of a “linker” holding theprotein to the membrane. While not wishing to be bound by theory, themolecules of the present invention may be used to exploit the cellulartrafficking described above. As such, in some embodiments of theinvention, polynucleotides, primary constructs or mmRNA are provided toexpress a secreted protein. The secreted proteins may be selected fromthose described herein or those in US Patent Publication, 20100255574,the contents of which are incorporated herein by reference in theirentirety.

In one embodiment, these may be used in the manufacture of largequantities of valuable human gene products.

Plasma Membrane Proteins

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a protein of the plasmamembrane.

Cytoplasmic or Cytoskeletal Proteins

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a cytoplasmic orcytoskeletal protein.

Intracellular Membrane Bound Proteins

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express an intracellular membranebound protein.

Nuclear Proteins

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a nuclear protein.

Proteins Associated with Human Disease

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a protein associated withhuman disease.

Miscellaneous Proteins

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a protein with a presentlyunknown therapeutic function.

Targeting Moieties

In some embodiments of the invention, polynucleotides, primaryconstructs or mmRNA are provided to express a targeting moiety. Theseinclude a protein-binding partner or a receptor on the surface of thecell, which functions to target the cell to a specific tissue space orto interact with a specific moiety, either in vivo or in vitro. Suitableprotein-binding partners include, but are not limited to, antibodies andfunctional fragments thereof, scaffold proteins, or peptides.Additionally, polynucleotide, primary construct or mmRNA can be employedto direct the synthesis and extracellular localization of lipids,carbohydrates, or other biological moieties or biomolecules.

Polypeptide Libraries

In one embodiment, the polynucleotides, primary constructs or mmRNA maybe used to produce polypeptide libraries. These libraries may arise fromthe production of a population of polynucleotides, primary constructs ormmRNA, each containing various structural or chemical modificationdesigns. In this embodiment, a population of polynucleotides, primaryconstructs or mmRNA may comprise a plurality of encoded polypeptides,including but not limited to, an antibody or antibody fragment, proteinbinding partner, scaffold protein, and other polypeptides taught hereinor known in the art. In a preferred embodiment, the polynucleotides areprimary constructs of the present invention, including mmRNA which maybe suitable for direct introduction into a target cell or culture whichin turn may synthesize the encoded polypeptides.

In certain embodiments, multiple variants of a protein, each withdifferent amino acid modification(s), may be produced and tested todetermine the best variant in terms of pharmacokinetics, stability,biocompatibility, and/or biological activity, or a biophysical propertysuch as expression level. Such a library may contain 10, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or over 10⁹ possible variants (including, butnot limited to, substitutions, deletions of one or more residues, andinsertion of one or more residues).

Anti-Microbial and Anti-Viral Polypeptides

The polynucleotides, primary constructs and mmRNA of the presentinvention may be designed to encode on or more antimicrobial peptides(AMP) or antiviral peptides (AVP). AMPs and AVPs have been isolated anddescribed from a wide range of animals such as, but not limited to,microorganisms, invertebrates, plants, amphibians, birds, fish, andmammals (Wang et al., Nucleic Acids Res. 2009; 37 (Databaseissue):D933-7). For example, anti-microbial polypeptides are describedin Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php; Wanget al., Nucleic Acids Res. 2009; 37 (Database issue):D933-7), CAMP:Collection of Anti-Microbial Peptides(http://www.bicnirrh.res.in/antimicrobial/); Thomas et al., NucleicAcids Res. 2010; 38 (Database issue):D774-80), U.S. Pat. No. 5,221,732,U.S. Pat. No. 5,447,914, U.S. Pat. No. 5,519,115, U.S. Pat. No.5,607,914, U.S. Pat. No. 5,714,577, U.S. Pat. No. 5,734,015, U.S. Pat.No. 5,798,336, U.S. Pat. No. 5,821,224, U.S. Pat. No. 5,849,490, U.S.Pat. No. 5,856,127, U.S. Pat. No. 5,905,187, U.S. Pat. No. 5,994,308,U.S. Pat. No. 5,998,374, U.S. Pat. No. 6,107,460, U.S. Pat. No.6,191,254, U.S. Pat. No. 6,211,148, U.S. Pat. No. 6,300,489, U.S. Pat.No. 6,329,504, U.S. Pat. No. 6,399,370, U.S. Pat. No. 6,476,189, U.S.Pat. No. 6,478,825, U.S. Pat. No. 6,492,328, U.S. Pat. No. 6,514,701,U.S. Pat. No. 6,573,361, U.S. Pat. No. 6,573,361, U.S. Pat. No.6,576,755, U.S. Pat. No. 6,605,698, U.S. Pat. No. 6,624,140, U.S. Pat.No. 6,638,531, U.S. Pat. No. 6,642,203, U.S. Pat. No. 6,653,280, U.S.Pat. No. 6,696,238, U.S. Pat. No. 6,727,066, U.S. Pat. No. 6,730,659,U.S. Pat. No. 6,743,598, U.S. Pat. No. 6,743,769, U.S. Pat. No.6,747,007, U.S. Pat. No. 6,790,833, U.S. Pat. No. 6,794,490, U.S. Pat.No. 6,818,407, U.S. Pat. No. 6,835,536, U.S. Pat. No. 6,835,713, U.S.Pat. No. 6,838,435, U.S. Pat. No. 6,872,705, U.S. Pat. No. 6,875,907,U.S. Pat. No. 6,884,776, U.S. Pat. No. 6,887,847, U.S. Pat. No.6,906,035, U.S. Pat. No. 6,911,524, U.S. Pat. No. 6,936,432, U.S. Pat.No. 7,001,924, U.S. Pat. No. 7,071,293, U.S. Pat. No. 7,078,380, U.S.Pat. No. 7,091,185, U.S. Pat. No. 7,094,759, U.S. Pat. No. 7,166,769,U.S. Pat. No. 7,244,710, U.S. Pat. No. 7,314,858, and U.S. Pat. No.7,582,301, the contents of which are incorporated by reference in theirentirety.

The anti-microbial polypeptides described herein may block cell fusionand/or viral entry by one or more enveloped viruses (e.g., HIV, HCV).For example, the anti-microbial polypeptide can comprise or consist of asynthetic peptide corresponding to a region, e.g., a consecutivesequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or60 amino acids of the transmembrane subunit of a viral envelope protein,e.g., HIV-1 gp120 or gp41. The amino acid and nucleotide sequences ofHIV-1 gp120 or gp41 are described in, e.g., Kuiken et al., (2008). “HIVSequence Compendium,” Los Alamos National Laboratory.

In some embodiments, the anti-microbial polypeptide may have at leastabout 75%, 80%, 85%, 90%, 95%, 100% sequence homology to thecorresponding viral protein sequence. In some embodiments, theanti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%,95%, or 100% sequence homology to the corresponding viral proteinsequence.

In other embodiments, the anti-microbial polypeptide may comprise orconsist of a synthetic peptide corresponding to a region, e.g., aconsecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, or 60 amino acids of the binding domain of a capsid bindingprotein. In some embodiments, the anti-microbial polypeptide may have atleast about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to thecorresponding sequence of the capsid binding protein.

The anti-microbial polypeptides described herein may block proteasedimerization and inhibit cleavage of viral proproteins (e.g., HIVGag-pol processing) into functional proteins thereby preventing releaseof one or more enveloped viruses (e.g., HIV, HCV). In some embodiments,the anti-microbial polypeptide may have at least about 75%, 80%, 85%,90%, 95%, 100% sequence homology to the corresponding viral proteinsequence.

In other embodiments, the anti-microbial polypeptide can comprise orconsist of a synthetic peptide corresponding to a region, e.g., aconsecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, or 60 amino acids of the binding domain of a proteasebinding protein. In some embodiments, the anti-microbial polypeptide mayhave at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology tothe corresponding sequence of the protease binding protein.

The anti-microbial polypeptides described herein can include an invitro-evolved polypeptide directed against a viral pathogen.

Anti-Microbial Polypeptides

Anti-microbial polypeptides (AMPs) are small peptides of variablelength, sequence and structure with broad spectrum activity against awide range of microorganisms including, but not limited to, bacteria,viruses, fungi, protozoa, parasites, prions, and tumor/cancer cells.(See, e.g., Zaiou, J Mol Med, 2007; 85:317; herein incorporated byreference in its entirety). It has been shown that AMPs havebroad-spectrum of rapid onset of killing activities, with potentiallylow levels of induced resistance and concomitant broad anti-inflammatoryeffects.

In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be under 10 kDa, e.g., under 8 kDa, 6kDa, 4 kDa, 2 kDa, or 1 kDa. In some embodiments, the anti-microbialpolypeptide (e.g., an anti-bacterial polypeptide) consists of from about6 to about 100 amino acids, e.g., from about 6 to about 75 amino acids,about 6 to about 50 amino acids, about 6 to about 25 amino acids, about25 to about 100 amino acids, about 50 to about 100 amino acids, or about75 to about 100 amino acids. In certain embodiments, the anti-microbialpolypeptide (e.g., an anti-bacterial polypeptide) may consist of fromabout 15 to about 45 amino acids. In some embodiments, theanti-microbial polypeptide (e.g., an anti-bacterial polypeptide) issubstantially cationic.

In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be substantially amphipathic. In certainembodiments, the anti-microbial polypeptide (e.g., an anti-bacterialpolypeptide) may be substantially cationic and amphipathic. In someembodiments, the anti-microbial polypeptide (e.g., an anti-bacterialpolypeptide) may be cytostatic to a Gram-positive bacterium. In someembodiments, the anti-microbial polypeptide (e.g., an anti-bacterialpolypeptide) may be cytotoxic to a Gram-positive bacterium. In someembodiments, the anti-microbial polypeptide (e.g., an anti-bacterialpolypeptide) may be cytostatic and cytotoxic to a Gram-positivebacterium. In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be cytostatic to a Gram-negativebacterium. In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be cytotoxic to a Gram-negativebacterium. In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be cytostatic and cytotoxic to aGram-positive bacterium. In some embodiments, the anti-microbialpolypeptide may be cytostatic to a virus, fungus, protozoan, parasite,prion, or a combination thereof. In some embodiments, the anti-microbialpolypeptide may be cytotoxic to a virus, fungus, protozoan, parasite,prion, or a combination thereof. In certain embodiments, theanti-microbial polypeptide may be cytostatic and cytotoxic to a virus,fungus, protozoan, parasite, prion, or a combination thereof. In someembodiments, the anti-microbial polypeptide may be cytotoxic to a tumoror cancer cell (e.g., a human tumor and/or cancer cell). In someembodiments, the anti-microbial polypeptide may be cytostatic to a tumoror cancer cell (e.g., a human tumor and/or cancer cell). In certainembodiments, the anti-microbial polypeptide may be cytotoxic andcytostatic to a tumor or cancer cell (e.g., a human tumor or cancercell). In some embodiments, the anti-microbial polypeptide (e.g., ananti-bacterial polypeptide) may be a secreted polypeptide.

In some embodiments, the anti-microbial polypeptide comprises orconsists of a defensin. Exemplary defensins include, but are not limitedto, α-defensins (e.g., neutrophil defensin 1, defensin alpha 1,neutrophil defensin 3, neutrophil defensin 4, defensin 5, defensin 6),β-defensins (e.g., beta-defensin 1, beta-defensin 2, beta-defensin 103,beta-defensin 107, beta-defensin 110, beta-defensin 136), andθ-defensins. In other embodiments, the anti-microbial polypeptidecomprises or consists of a cathelicidin (e.g., hCAP18).

Anti-Viral Polypeptides

Anti-viral polypeptides (AVPs) are small peptides of variable length,sequence and structure with broad spectrum activity against a wide rangeof viruses. See, e.g., Zaiou, J Mol Med, 2007; 85:317. It has been shownthat AVPs have a broad-spectrum of rapid onset of killing activities,with potentially low levels of induced resistance and concomitant broadanti-inflammatory effects. In some embodiments, the anti-viralpolypeptide is under 10 kDa, e.g., under 8 kDa, 6 kDa, 4 kDa, 2 kDa, or1 kDa. In some embodiments, the anti-viral polypeptide comprises orconsists of from about 6 to about 100 amino acids, e.g., from about 6 toabout 75 amino acids, about 6 to about 50 amino acids, about 6 to about25 amino acids, about 25 to about 100 amino acids, about 50 to about 100amino acids, or about 75 to about 100 amino acids. In certainembodiments, the anti-viral polypeptide comprises or consists of fromabout 15 to about 45 amino acids. In some embodiments, the anti-viralpolypeptide is substantially cationic. In some embodiments, theanti-viral polypeptide is substantially amphipathic. In certainembodiments, the anti-viral polypeptide is substantially cationic andamphipathic. In some embodiments, the anti-viral polypeptide iscytostatic to a virus. In some embodiments, the anti-viral polypeptideis cytotoxic to a virus. In some embodiments, the anti-viral polypeptideis cytostatic and cytotoxic to a virus. In some embodiments, theanti-viral polypeptide is cytostatic to a bacterium, fungus, protozoan,parasite, prion, or a combination thereof. In some embodiments, theanti-viral polypeptide is cytotoxic to a bacterium, fungus, protozoan,parasite, prion or a combination thereof. In certain embodiments, theanti-viral polypeptide is cytostatic and cytotoxic to a bacterium,fungus, protozoan, parasite, prion, or a combination thereof. In someembodiments, the anti-viral polypeptide is cytotoxic to a tumor orcancer cell (e.g., a human cancer cell). In some embodiments, theanti-viral polypeptide is cytostatic to a tumor or cancer cell (e.g., ahuman cancer cell). In certain embodiments, the anti-viral polypeptideis cytotoxic and cytostatic to a tumor or cancer cell (e.g., a humancancer cell). In some embodiments, the anti-viral polypeptide is asecreted polypeptide.

Cytotoxic Nucleosides

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe present invention may incorporate one or more cytotoxic nucleosides.Further, novel enzyme or enzyme variants such as RNA polymerases may beevolved which are permissive to the incorporation of cytotoxicchemically modified NTPs. For example, cytotoxic nucleosides may beincorporated into polynucleotides, primary constructs or mmRNA such asbifunctional modified RNAs or mRNAs. Cytotoxic nucleoside anti-canceragents include, but are not limited to, adenosine arabinoside,cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine,floxuridine, FTORAFUR® (a combination of tegafur and uracil), tegafur((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), and6-mercaptopurine.

A number of cytotoxic nucleoside analogues are in clinical use, or havebeen the subject of clinical trials, as anticancer agents. Examples ofsuch analogues include, but are not limited to, cytarabine, gemcitabine,troxacitabine, decitabine, tezacitabine, 2′-deoxy-2′-methylidenecytidine(DMDC), cladribine, clofarabine, 5-azacytidine, 4′-thio-aracytidine,cyclopentenylcytosine and1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine. Anotherexample of such a compound is fludarabine phosphate. These compounds maybe administered systemically and may have side effects which are typicalof cytotoxic agents such as, but not limited to, little or nospecificity for tumor cells over proliferating normal cells.

A number of prodrugs of cytotoxic nucleoside analogues are also reportedin the art. Examples include, but are not limited to,N4-behenoyl-1-beta-D-arabinofuranosylcytosine,N4-octadecyl-1-beta-D-arabinofuranosylcytosine,N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5′-elaidic acid ester). In general,these prodrugs may be converted into the active drugs mainly in theliver and systemic circulation and display little or no selectiverelease of active drug in the tumor tissue. For example, capecitabine, aprodrug of 5′-deoxy-5-fluorocytidine (and eventually of 5-fluorouracil),is metabolized both in the liver and in the tumor tissue. A series ofcapecitabine analogues containing “an easily hydrolysable radical underphysiological conditions” has been claimed by Fujiu et al. (U.S. Pat.No. 4,966,891) and is herein incorporated by reference. The seriesdescribed by Fujiu includes N4 alkyl and aralkyl carbamates of5′-deoxy-5-fluorocytidine and the implication that these compounds willbe activated by hydrolysis under normal physiological conditions toprovide 5′-deoxy-5-fluorocytidine.

A series of cytarabine N4-carbamates has been by reported by Fadl et al(Pharmazie. 1995, 50, 382-7, herein incorporated by reference) in whichcompounds were designed to convert into cytarabine in the liver andplasma. WO 2004/041203, herein incorporated by reference, disclosesprodrugs of gemcitabine, where some of the prodrugs are N4-carbamates.These compounds were designed to overcome the gastrointestinal toxicityof gemcitabine and were intended to provide gemcitabine by hydrolyticrelease in the liver and plasma after absorption of the intact prodrugfrom the gastrointestinal tract. Nomura et al (Bioorg Med. Chem. 2003,11, 2453-61, herein incorporated by reference) have described acetalderivatives of 1-(3-C-ethynyl-β-D-ribo-pentofaranosyl) cytosine which,on bioreduction, produced an intermediate that required furtherhydrolysis under acidic conditions to produce a cytotoxic nucleosidecompound.

Cytotoxic nucleotides which may be chemotherapeutic also include, butare not limited to, pyrazolo[3,4-D]-pyrimidines, allopurinol,azathioprine, capecitabine, cytosine arabinoside, fluorouracil,mercaptopurine, 6-thioguanine, acyclovir, ara-adenosine, ribavirin,7-deaza-adenosine, 7-deaza-guanosine, 6-aza-uracil, 6-aza-cytidine,thymidine ribonucleotide, 5-bromodeoxyuridine, 2-chloro-purine, andinosine, or combinations thereof.

Flanking Regions: Untranslated Regions (UTRs)

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon;whereas, the 3′UTR starts immediately following the stop codon andcontinues until the transcriptional termination signal. There is growingbody of evidence about the regulatory roles played by the UTRs in termsof stability of the nucleic acid molecule and translation. Theregulatory features of a UTR can be incorporated into thepolynucleotides, primary constructs and/or mmRNA of the presentinvention to enhance the stability of the molecule. The specificfeatures can also be incorporated to ensure controlled down-regulationof the transcript in case they are misdirected to undesired organssites. It is also contemplated by the present invention, that novelenzymes and/or enzyme variants may be utilized to incorporate chemicallymodified NTPs in unique regions of the primary construct, including butnot limited to flanking regions such as the UTRs.

5′ UTR and Translation Initiation

Natural 5′UTRs bear features which play roles in for translationinitiation. They harbor signatures like Kozak sequences which arecommonly known to be involved in the process by which the ribosomeinitiates translation of many genes. Kozak sequences have the consensusCCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three basesupstream of the start codon (AUG), which is followed by another ‘G’.5′UTR also have been known to form secondary structures which areinvolved in elongation factor binding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of the polynucleotides, primary constructs or mmRNAof the invention. For example, introduction of 5′ UTR of liver-expressedmRNA, such as albumin, serum amyloid A, Apolipoprotein A/B/E,transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, could beused to enhance expression of a nucleic acid molecule, such as a mmRNA,in hepatic cell lines or liver. Likewise, use of 5′ UTR from othertissue-specific mRNA to improve expression in that tissue ispossible—for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), forendothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF,GM-CSF, CD11b, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), foradipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lungepithelial cells (SP-A/B/C/D).

Other non-UTR sequences may be incorporated into the 5′ (or 3′ UTR)UTRs. For example, introns or portions of introns sequences may beincorporated into the flanking regions of the polynucleotides, primaryconstructs or mmRNA of the invention. Incorporation of intronicsequences may increase protein production as well as mRNA levels.

The 5′UTR may selected for use in the present invention may be astructured UTR such as, but not limited to, 5′UTRs to controltranslation. As a non-limiting example, a structured 5′UTR may bebeneficial when using any of the terminal modifications described incopending U.S. Provisional Application No. 61,758,921 filed Jan. 31,2013, entitled Differential Targeting Using RNA Constructs; U.S.Provisional Application No. 61/781,139 filed Mar. 14, 2013, entitledDifferential Targeting Using RNA Constructs; U.S. ProvisionalApplication No. 61/729,933, filed Nov. 26, 2012 entitled TerminallyOptimized RNAs and U.S. Provisional Application No. 61/737,224 filedDec. 14, 2012 entitled Terminally Optimized RNAs; each of which isherein incorporated by reference in their entirety.

Incorporating microRNA Binding Sites

In one embodiment modified nucleic acids (mRNA), enhanced modified RNAor ribonucleic acids of the invention would not only encode apolypeptide but also a sensor sequence. Sensor sequences include, forexample, microRNA binding sites, transcription factor binding sites,artificial binding sites engineered to act as pseudo-receptors forendogenous nucleic acid binding molecules.

In one embodiment, microRNA (miRNA) profiling of the target cells ortissues is conducted to determine the presence or absence of miRNA inthe cells or tissues.

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′UTR of nucleic acid molecules and down-regulate gene expressioneither by reducing nucleic acid molecule stability or by inhibitingtranslation. The modified nucleic acids (mRNA), enhanced modified RNA orribonucleic acids of the invention may comprise one or more microRNAtarget sequences, microRNA sequences, or microRNA seeds. Such sequencesmay correspond to any known microRNA such as those taught in USPublication US2005/0261218 and US Publication US2005/0059005, thecontents of which are incorporated herein by reference in theirentirety.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked by an adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. The bases of themicroRNA seed have complete complementarity with the target sequence. Byengineering microRNA target sequences into the 3′UTR of nucleic acids ormRNA of the invention one can target the molecule for degradation orreduced translation, provided the microRNA in question is available.This process will reduce the hazard of off target effects upon nucleicacid molecule delivery. Identification of microRNA, microRNA targetregions, and their expression patterns and role in biology have beenreported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand andCheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia2012 26:404-413 (2011 Dec. 20. doi: 10.1038/1eu.2011.356); Bartel Cell2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner andNaldini, Tissue Antigens. 2012 80:393-403 and all references therein;each of which is herein incorporated by reference in its entirety).

For example, if the mRNA is not intended to be delivered to the liverbut ends up there, then miR-122, a microRNA abundant in liver, caninhibit the expression of the gene of interest if one or multiple targetsites of miR-122 are engineered into the 3′UTR of the modified nucleicacids, enhanced modified RNA or ribonucleic acids. Introduction of oneor multiple binding sites for different microRNA can be engineered tofurther decrease the longevity, stability, and protein translation of amodified nucleic acids, enhanced modified RNA or ribonucleic acids. Asused herein, the term “microRNA site” refers to a microRNA target siteor a microRNA recognition site, or any nucleotide sequence to which amicroRNA binds or associates. It should be understood that “binding” mayfollow traditional Watson-Crick hybridization rules or may reflect anystable association of the microRNA with the target sequence at oradjacent to the microRNA site.

Conversely, for the purposes of the modified nucleic acids, enhancedmodified RNA or ribonucleic acids of the present invention, microRNAbinding sites can be engineered out of (i.e. removed from) sequences inwhich they naturally occur in order to increase protein expression inspecific tissues. For example, miR-122 binding sites may be removed toimprove protein expression in the liver.

Regulation of expression in multiple tissues can be accomplished throughintroduction or removal or one or several microRNA binding sites.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-id, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126).

MicroRNA can also regulate complex biological processes such asangiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 201118:171-176). In the modified nucleic acids, enhanced modified RNA orribonucleic acids of the invention, binding sites for microRNAs that areinvolved in such processes may be removed or introduced, in order totailor the expression of the modified nucleic acids, enhanced modifiedRNA or ribonucleic acids expression to biologically relevant cell typesor to the context of relevant biological processes. In this context, themRNA are defined as auxotrophic mRNA.

At least one microRNA site can be engineered into the 3′ UTR of themodified nucleic acids, enhanced modified RNA or ribonucleic acids ofthe present invention. In this context, at least two, at least three, atleast four, at least five, at least six, at least seven, at least eight,at least nine, at least ten or more microRNA sites may be engineeredinto the 3′ UTR of the ribonucleic acids of the present invention. Inone embodiment, the microRNA sites incorporated into the modifiednucleic acids, enhanced modified RNA or ribonucleic acids may be thesame or may be different microRNA sites. In another embodiment, themicroRNA sites incorporated into the modified nucleic acids, enhancedmodified RNA or ribonucleic acids may target the same or differenttissues in the body. As a non-limiting example, through the introductionof tissue-, cell-type-, or disease-specific microRNA binding sites inthe 3′ UTR of a modified nucleic acid mRNA, the degree of expression inspecific cell types (e.g. hepatocytes, myeloid cells, endothelial cells,cancer cells, etc.) can be reduced.

In one embodiment, a nucleic acid may be engineered to include microRNAsites which are expressed in different tissues of a subject. As anon-limiting example, a modified nucleic acid, enhanced modified RNA orribonucleic acid of the present invention may be engineered to includemiR-192 and miR-122 to regulate expression of the modified nucleic acid,enhanced modified RNA or ribonucleic acid in the liver and kidneys of asubject. In another embodiment, a modified nucleic acid, enhancedmodified RNA or ribonucleic acid may be engineered to include more thanone microRNA sites for the same tissue. For example, a modified nucleicacid, enhanced modified RNA or ribonucleic acid of the present inventionmay be engineered to include miR-17-92 and miR-126 to regulateexpression of the modified nucleic acid, enhanced modified RNA orribonucleic acid in endothelial cells of a subject.

In one embodiment, the therapeutic window and or differential expressionassociated with the target polypeptide encoded by the modified nucleicacid, enhanced modified RNA or ribonucleic acid encoding a signal (alsoreferred to herein as a polynucleotide) of the invention may be altered.For example, polynucleotides may be designed whereby a death signal ismore highly expressed in cancer cells (or a survival signal in a normalcell) by virtue of the miRNA signature of those cells. Where a cancercell expresses a lower level of a particular miRNA, the polynucleotideencoding the binding site for that miRNA (or miRNAs) would be morehighly expressed. Hence, the target polypeptide encoded by thepolynucleotide is selected as a protein which triggers or induces celldeath. Neigboring noncancer cells, harboring a higher expression of thesame miRNA would be less affected by the encoded death signal as thepolynucleotide would be expressed at a lower level due to the affects ofthe miRNA binding to the binding site or “sensor” encoded in the 3′UTR.Conversely, cell survival or cytoprotective signals may be delivered totissues containing cancer and non cancerous cells where a miRNA has ahigher expression in the cancer cells—the result being a lower survivalsignal to the cancer cell and a larger survival signature to the normalcell. Multiple polynucleotides may be designed and administered havingdifferent signals according to the previous paradigm.

According to the present invention, the polynucleotides may be modifiedas to avoid the deficiencies of other polypeptide-encoding molecules ofthe art. Hence, in this embodiment the polynucleotides are referred toas modified polynucleotides.

Through an understanding of the expression patterns of microRNA indifferent cell types, modified nucleic acids, enhanced modified RNA orribonucleic acids such as polynucleotides can be engineered for moretargeted expression in specific cell types or only under specificbiological conditions. Through introduction of tissue-specific microRNAbinding sites, modified nucleic acids, enhanced modified RNA orribonucleic acids, could be designed that would be optimal for proteinexpression in a tissue or in the context of a biological condition.

Transfection experiments can be conducted in relevant cell lines, usingengineered modified nucleic acids, enhanced modified RNA or ribonucleicacids and protein production can be assayed at various time pointspost-transfection. For example, cells can be transfected with differentmicroRNA binding site-engineering nucleic acids or mRNA and by using anELISA kit to the relevant protein and assaying protein produced at 6 hr,12 hr, 24 hr, 48 hr, 72 hr and 7 days post-transfection. In vivoexperiments can also be conducted using microRNA-binding site-engineeredmolecules to examine changes in tissue-specific expression of formulatedmodified nucleic acids, enhanced modified RNA or ribonucleic acids.

3′ UTR and the AU Rich Elements

3′UTRs are known to have stretches of Adenosines and Uridines embeddedin them. These AU rich signatures are particularly prevalent in geneswith high rates of turnover. Based on their sequence features andfunctional properties, the AU rich elements (AREs) can be separated intothree classes (Chen et al, 1995): Class I AREs contain several dispersedcopies of an AUUUA motif within U-rich regions. C-Myc and MyoD containclass I AREs. Class II AREs possess two or more overlappingUUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREsinclude GM-CSF and TNF-a. Class III ARES are less well defined. These Urich regions do not contain an AUUUA motif. c-Jun and Myogenin are twowell-studied examples of this class. Most proteins binding to the AREsare known to destabilize the messenger, whereas members of the ELAVfamily, most notably HuR, have been documented to increase the stabilityof mRNA. HuR binds to AREs of all the three classes. Engineering the HuRspecific binding sites into the 3′ UTR of nucleic acid molecules willlead to HuR binding and thus, stabilization of the message in vivo.

Introduction, removal or modification of 3′ UTR AU rich elements (AREs)can be used to modulate the stability of polynucleotides, primaryconstructs or mmRNA of the invention. When engineering specificpolynucleotides, primary constructs or mmRNA, one or more copies of anARE can be introduced to make polynucleotides, primary constructs ormmRNA of the invention less stable and thereby curtail translation anddecrease production of the resultant protein. Likewise, AREs can beidentified and removed or mutated to increase the intracellularstability and thus increase translation and production of the resultantprotein. Transfection experiments can be conducted in relevant celllines, using polynucleotides, primary constructs or mmRNA of theinvention and protein production can be assayed at various time pointspost-transfection. For example, cells can be transfected with differentARE-engineering molecules and by using an ELISA kit to the relevantprotein and assaying protein produced at 6 hr, 12 hr, 24 hr, 48 hr, and7 days post-transfection.

Incorporating microRNA Binding Sites

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′UTR of nucleic acid molecules and down-regulate gene expressioneither by reducing nucleic acid molecule stability or by inhibitingtranslation. The polynucleotides, primary constructs or mmRNA of theinvention may comprise one or more microRNA target sequences, microRNAsequences, or microRNA seeds. Such sequences may correspond to any knownmicroRNA such as those taught in US Publication US2005/0261218 and USPublication US2005/0059005, the contents of which are incorporatedherein by reference in their entirety.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked byan adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. The bases of themicroRNA seed have complete complementarity with the target sequence. Byengineering microRNA target sequences into the 3′UTR of polynucleotides,primary constructs or mmRNA of the invention one can target the moleculefor degradation or reduced translation, provided the microRNA inquestion is available. This process will reduce the hazard of off targeteffects upon nucleic acid molecule delivery. Identification of microRNA,microRNA target regions, and their expression patterns and role inbiology have been reported (Bonauer et al., Curr Drug Targets 201011:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176;Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20. doi:10.1038/1eu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al,Cell, 2007 129:1401-1414).

For example, if the nucleic acid molecule is an mRNA and is not intendedto be delivered to the liver but ends up there, then miR-122, a microRNAabundant in liver, can inhibit the expression of the gene of interest ifone or multiple target sites of miR-122 are engineered into the 3′UTR ofthe polynucleotides, primary constructs or mmRNA. Introduction of one ormultiple binding sites for different microRNA can be engineered tofurther decrease the longevity, stability, and protein translation of apolynucleotides, primary constructs or mmRNA.

As used herein, the term “microRNA site” refers to a microRNA targetsite or a microRNA recognition site, or any nucleotide sequence to whicha microRNA binds or associates. It should be understood that “binding”may follow traditional Watson-Crick hybridization rules or may reflectany stable association of the microRNA with the target sequence at oradjacent to the microRNA site.

Conversely, for the purposes of the polynucleotides, primary constructsor mmRNA of the present invention, microRNA binding sites can beengineered out of (i.e. removed from) sequences in which they naturallyoccur in order to increase protein expression in specific tissues. Forexample, miR-122 binding sites may be removed to improve proteinexpression in the liver. Regulation of expression in multiple tissuescan be accomplished through introduction or removal or one or severalmicroRNA binding sites.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-id, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulatecomplex biological processes such as angiogenesis (miR-132) (Anand andCheresh Curr Opin Hematol 2011 18:171-176). In polynucleotides, primaryconstructs or mmRNA of the invention, binding sites for microRNAs thatare involved in such processes may be removed or introduced, in order totailor the expression of the polynucleotides, primary constructs ormmRNA expression to biologically relevant cell types or to the contextof relevant biological processes.

Lastly, through an understanding of the expression patterns of microRNAin different cell types, polynucleotides, primary constructs or mmRNAcan be engineered for more targeted expression in specific cell types oronly under specific biological conditions. Through introduction oftissue-specific microRNA binding sites, polynucleotides, primaryconstructs or mmRNA could be designed that would be optimal for proteinexpression in a tissue or in the context of a biological condition.

Transfection experiments can be conducted in relevant cell lines, usingengineered polynucleotides, primary constructs or mmRNA and proteinproduction can be assayed at various time points post-transfection. Forexample, cells can be transfected with different microRNA bindingsite-engineering polynucleotides, primary constructs or mmRNA and byusing an ELISA kit to the relevant protein and assaying protein producedat 6 hr, 12 hr, 24 hr, 48 hr, 72 hr and 7 days post-transfection. Invivo experiments can also be conducted using microRNA-bindingsite-engineered molecules to examine changes in tissue-specificexpression of formulated polynucleotides, primary constructs or mmRNA.

5′ Capping

The 5′ cap structure of an mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsibile for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

Modifications to the polynucleotides, primary constructs, and mmRNA ofthe present invention may generate a non-hydrolyzable cap structurepreventing decapping and thus increasing mRNA half-life. Because capstructure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiesterlinkages, modified nucleotides may be used during the capping reaction.For example, a Vaccinia Capping Enzyme from New England Biolabs(Ipswich, Mass.) may be used with α-thio-guanosine nucleotides accordingto the manufacturer's instructions to create a phosphorothioate linkagein the 5′-ppp-5′ cap. Additional modified guanosine nucleotides may beused such as α-methyl-phosphonate and seleno-phosphate nucleotides.Furthermore, novel capping enzymes may be evolved which are permissiveto various cap structures or other chemically modified NTPs whichfunction as caps for RNA or mRNA.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the mRNA (as mentioned above) on the2′-hydroxyl group of the sugar ring. Multiple distinct 5′-cap structurescan be used to generate the 5′-cap of a nucleic acid molecule, such asan mRNA molecule. Novel transferase or methylase enzymes may be evolvedvia PACE which effect the addition or removal of one or more methyl,ethyl, carboxy groups to the RNA or mRNA.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e. endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e. non-enzymatically) orenzymatically synthesized and/linked to a nucleic acid molecule.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′mppp-G;which may equivaliently be designated 3′ 0-Me-m7G(5)ppp(5′)G). The 3′-Oatom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA).The N7- and 3′-O-methlyated guanine provides the terminal moiety of thecapped nucleic acid molecule (e.g. mRNA or mmRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

Yet another exemplary cap analog for use in the present invention is onethat contains a N⁷⁻(4-chlorophenoxyethyl) moiety such as, but notlimited to, N⁷⁻(4-chlorophenoxyethyl)-G-(5′)ppp(5′)G orN⁷⁻(4-chlorophenoxyethyl)-m^(3′-O)G(5′)ppp(5′)G (Kore et al. Bioorganic& Medicinal Chemistry 21 (2013) 4570-4574; the contents of which areherein incorporated by reference in its entirety). As described by Koreet al, a cap comprising a N⁷⁻(4-chlorophenoxyethyl) moiety may bebeneficial over a standard mCAP analog as the 3′OH of either the G orm⁷G of the mCAP analog can serve as the initiating nucleophile fortranscriptional elongation which can lead to the synthesis of twoisomeric RNAs of forward or reverse form. Additionally, Kore et al.showed that both N⁷⁻(4-chlorophenoxyethyl)-G-(5′) ppp(5′)G andN⁷⁻(4-chlorophenoxyethyl)-m^(3′-O) G(5′)ppp(5′)G were a substrate for T7RNA polymerase. (Kore et al. Bioorganic & Medicinal Chemistry 21 (2013)4570-4574; the contents of which are herein incorporated by reference inits entirety).

While cap analogs allow for the concomitant capping of a nucleic acidmolecule in an in vitro transcription reaction, up to 20% of transcriptsremain uncapped. This, as well as the structural differences of a capanalog from an endogenous 5′-cap structures of nucleic acids produced bythe endogenous, cellular transcription machinery, may lead to reducedtranslational competency and reduced cellular stability.

Polynucleotides, primary constructs and mmRNA of the invention may alsobe capped post-transcriptionally, using enzymes, in order to generatemore authentic 5′-cap structures. As used herein, the phrase “moreauthentic” refers to a feature that closely mirrors or mimics, eitherstructurally or functionally, an endogenous or wild type feature. Thatis, a “more authentic” feature is better representative of anendogenous, wild-type, natural or physiological cellular function and/orstructure as compared to synthetic features or analogs, etc., of theprior art, or which outperforms the corresponding endogenous, wild-type,natural or physiological feature in one or more respects. Non-limitingexamples of more authentic 5′cap structures of the present invention arethose which, among other things, have enhanced binding of cap bindingproteins, increased half life, reduced susceptibility to 5′endonucleases and/or reduced 5′decapping, as compared to synthetic 5′capstructures known in the art (or to a wild-type, natural or physiological5′cap structure). For example, recombinant Vaccinia Virus Capping Enzymeand recombinant 2′-O-methyltransferase enzyme can create a canonical5′-5′-triphosphate linkage between the 5′-terminal nucleotide of an mRNAand a guanine cap nucleotide wherein the cap guanine contains an N7methylation and the 5′-terminal nucleotide of the mRNA contains a2′-O-methyl. Such a structure is termed the Cap1 structure. This capresults in a higher translational-competency and cellular stability anda reduced activation of cellular pro-inflammatory cytokines, ascompared, e.g., to other 5′cap analog structures known in the art. Capstructures include 7mG(5′)ppp(5′)N,pN2p (cap 0), 7mG(5′)ppp(5′)NlmpNp(cap 1), and 7mG(5′)-ppp(5′)NlmpN2mp (cap 2). It is contemplated by thepresent invention that variants of either the vaccinia capping enzyme orthe O-methyltransferase may be evolved to more efficiently cap RNAmolecules or mmRNA of the invention.

Because the polynucleotides, primary constructs or mmRNA may be cappedpost-transcriptionally, and because this process is more efficient,nearly 100% of the polynucleotides, primary constructs or mmRNA may becapped. This is in contrast to −80% when a cap analog is linked to anmRNA in the course of an in vitro transcription reaction.

According to the present invention, 5′ terminal caps may includeendogenous caps or cap analogs. According to the present invention, a 5′terminal cap may comprise a guanine analog. Useful guanine analogsinclude inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,and 2-azido-guanosine.

Viral Sequences

Additional viral sequences such as, but not limited to, the translationenhancer sequence of the barley yellow dwarf virus (BYDV-PAV) can beengineered and inserted in the 3′ UTR of the polynucleotides, primaryconstructs or mmRNA of the invention and can stimulate the translationof the construct in vitro and in vivo. Transfection experiments can beconducted in relevant cell lines at and protein production can beassayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day 7post-transfection.

IRES Sequences

Further, provided are polynucleotides, primary constructs or mmRNA whichmay contain an internal ribosome entry site (IRES). First identified asa feature Picorna virus RNA, IRES plays an important role in initiatingprotein synthesis in absence of the 5′ cap structure. An IRES may act asthe sole ribosome binding site, or may serve as one of multiple ribosomebinding sites of an mRNA. Polynucleotides, primary constructs or mmRNAcontaining more than one functional ribosome binding site may encodeseveral peptides or polypeptides that are translated independently bythe ribosomes (“multicistronic nucleic acid molecules”). Whenpolynucleotides, primary constructs or mmRNA are provided with an IRES,further optionally provided is a second translatable region. Examples ofIRES sequences that can be used according to the invention includewithout limitation, those from picornaviruses (e.g. FMDV), pest viruses(CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV),foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV),classical swine fever viruses (CSFV), murine leukemia virus (MLV),simian immune deficiency viruses (SIV) or cricket paralysis viruses(CrPV).

Poly-A Tails

During RNA processing, a long chain of adenine nucleotides (poly-A tail)may be added to a polynucleotide such as an mRNA molecules in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript may be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between 100 and 250residues long.

It has been discovered that unique poly-A tail lengths provide certainadvantages to the polynucleotides, primary constructs or mmRNA of thepresent invention.

Generally, the length of a poly-A tail of the present invention isgreater than 30 nucleotides in length. In another embodiment, the poly-Atail is greater than 35 nucleotides in length (e.g., at least or greaterthan about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500,and 3,000 nucleotides). In some embodiments, the polynucleotide, primaryconstruct, or mmRNA includes from about 30 to about 3,000 nucleotides(e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500,from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000,from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500,from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000,from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

In one embodiment, the poly-A tail is designed relative to the length ofthe overall polynucleotides, primary constructs or mmRNA. This designmay be based on the length of the coding region, the length of aparticular feature or region (such as the first or flanking regions), orbased on the length of the ultimate product expressed from thepolynucleotides, primary constructs or mmRNA.

In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the polynucleotides, primaryconstructs or mmRNA or feature thereof. The poly-A tail may also bedesigned as a fraction of polynucleotides, primary constructs or mmRNAto which it belongs. In this context, the poly-A tail may be 10, 20, 30,40, 50, 60, 70, 80, or 90% or more of the total length of the constructor the total length of the construct minus the poly-A tail. Further,engineered binding sites and conjugation of polynucleotides, primaryconstructs or mmRNA for Poly-A binding protein may enhance expression.

Additionally, multiple distinct polynucleotides, primary constructs ormmRNA may be linked together to the PABP (Poly-A binding protein)through the 3′-end using modified nucleotides at the 3′-terminus of thepoly-A tail. Transfection experiments can be conducted in relevant celllines at and protein production can be assayed by ELISA at 12 hr, 24 hr,48 hr, 72 hr and day 7 post-transfection.

In one embodiment, the polynucleotide primary constructs of the presentinvention are designed to include a polyA-G Quartet. The G-quartet is acyclic hydrogen bonded array of four guanine nucleotides that can beformed by G-rich sequences in both DNA and RNA. In this embodiment, theG-quartet is incorporated at the end of the poly-A tail. The resultantmmRNA construct is assayed for stability, protein production and otherparameters including half-life at various time points. It has beendiscovered that the polyA-G quartet results in protein productionequivalent to at least 75% of that seen using a poly-A tail of 120nucleotides alone.

Quantification

In one embodiment, the polynucleotides, primary constructs or mmRNA ofthe present invention may be quantified in exosomes derived from one ormore bodily fluid. As used herein “bodily fluids” include peripheralblood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum,saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid,cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostaticfluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter,hair, tears, cyst fluid, pleural and peritoneal fluid, pericardialfluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus,sebum, vomit, vaginal secretions, mucosal secretion, stool water,pancreatic juice, lavage fluids from sinus cavities, bronchopulmonaryaspirates, blastocyl cavity fluid, and umbilical cord blood.Alternatively, exosomes may be retrieved from an organ selected from thegroup consisting of lung, heart, pancreas, stomach, intestine, bladder,kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus,liver, and placenta.

In the quantification method, a sample of not more than 2 mL is obtainedfrom the subject and the exosomes isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.In the analysis, the level or concentration of a polynucleotide, primaryconstruct or mmRNA may be an expression level, presence, absence,truncation or alteration of the administered construct. It isadvantageous to correlate the level with one or more clinical phenotypesor with an assay for a human disease biomarker. The assay may beperformed using construct specific probes, cytometry, qRT-PCR, real-timePCR, PCR, flow cytometry, electrophoresis, mass spectrometry, orcombinations thereof while the exosomes may be isolated usingimmunohistochemical methods such as enzyme linked immunosorbent assay(ELISA) methods. Exosomes may also be isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of polynucleotides, primary constructs or mmRNAremaining or delivered. This is possible because the polynucleotides,primary constructs or mmRNA of the present invention differ from theendogenous forms due to the structural or chemical modifications.

II. DESIGN AND SYNTHESIS OF MMRNA

Polynucleotides, primary constructs or mmRNA for use in accordance withthe invention may be prepared according to any available techniqueincluding, but not limited to chemical synthesis, enzymatic synthesis,which is generally termed in vitro transcription (IVT) or enzymatic orchemical cleavage of a longer precursor, etc. Methods of synthesizingRNAs are known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotidesynthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.:IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis:methods and applications, Methods in Molecular Biology, v. 288 (Clifton,N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporatedherein by reference).

The process of design and synthesis of the primary constructs of theinvention generally includes the steps of gene construction, mRNAproduction (either with or without modifications) and purification. Inthe enzymatic synthesis method, a target polynucleotide sequenceencoding the polypeptide of interest is first selected for incorporationinto a vector which will be amplified to produce a cDNA template.Optionally, the target polynucleotide sequence and/or any flankingsequences may be codon optimized. The cDNA template is then used toproduce mRNA through in vitro transcription (IVT). After production, themRNA may undergo purification and clean-up processes. The steps of whichare provided in more detail below.

Gene Construction

The step of gene construction may include, but is not limited to genesynthesis, vector amplification, plasmid purification, plasmidlinearization and clean-up, and cDNA template synthesis and clean-up.

Gene Synthesis

Once a polypeptide of interest, or target, is selected for production, aprimary construct is designed. Within the primary construct, a firstregion of linked nucleosides encoding the polypeptide of interest may beconstructed using an open reading frame (ORF) of a selected nucleic acid(DNA or RNA) transcript. The ORF may comprise the wild type ORF, anisoform, variant or a fragment thereof. As used herein, an “open readingframe” or “ORF” is meant to refer to a nucleic acid sequence (DNA orRNA) which is capable of encoding a polypeptide of interest. ORFs oftenbegin with the start codon, ATG and end with a nonsense or terminationcodon or signal.

Further, the nucleotide sequence of the first region may be codonoptimized. Codon optimization methods are known in the art and may beuseful in efforts to achieve one or more of several goals. These goalsinclude to match codon frequencies in target and host organisms toensure proper folding, bias GC content to increase mRNA stability orreduce secondary structures, minimize tandem repeat codons or base runsthat may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeprotein trafficking sequences, remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, to adjusttranslational rates to allow the various domains of the protein to foldproperly, or to reduce or eliminate problem secondary structures withinthe mRNA. Codon optimization tools, algorithms and services are known inthe art, non-limiting examples include services from GeneArt (LifeTechnologies) and/or DNA2.0 (Menlo Park Calif.). In one embodiment, theORF sequence is optimized using optimization algorithms. Codon optionsfor each amino acid are given in Table 1.

TABLE 1 Codon Options Single Letter Amino Acid Code Codon OptionsIsoleucine I ATT, ATC, ATA Leucine L CTT, CTC, CTA, CTG, TTA, TTG ValineV GTT, GTC, GTA, GTG Phenylalanine F TTT, TTC Methionine M ATG CysteineC TGT, TGC Alanine A GCT, GCC, GCA, GCG Glycine G GGT, GGC, GGA, GGGProline P CCT, CCC, CCA, CCG Threonine T ACT, ACC, ACA, ACG Serine STCT, TCC, TCA, TCG, AGT, AGC Tyrosine Y TAT, TAC Tryptophan W TGGGlutamine Q CAA, CAG Asparagine N AAT, AAC Histidine H CAT, CAC Glutamicacid E GAA, GAG Aspartic acid D GAT, GAC Lysine K AAA, AAG Arginine RCGT, CGC, CGA, CGG, AGA, AGG Selenocysteine Sec UGA in mRNA in presenceof Selenocystein insertion element (SECIS) Stop codons Stop TAA, TAG,TGA

In one embodiment, at least a portion of the polynucleotide sequence maybe codon optimized by methods known in the art and/or described herein.After a sequence has been codon optimized it may be further evaluatedfor regions containing restriction sites. At least one nucleotide withinthe restriction site regions may be replaced with another nucleotide inorder to remove the restriction site from the sequence but thereplacement of nucleotides does alter the amino acid sequence which isencoded by the codon optimized nucleotide sequence.

Features, which may be considered beneficial in some embodiments of thepresent invention, may be encoded by the primary construct and may flankthe ORF as a first or second flanking region. The flanking regions maybe incorporated into the primary construct before and/or afteroptimization of the ORF. It is not required that a primary constructcontain both a 5′ and 3′ flanking region. Examples of such featuresinclude, but are not limited to, untranslated regions (UTRs), Kozaksequences, an oligo(dT) sequence, and detectable tags and may includemultiple cloning sites which may have XbaI recognition.

In some embodiments, a 5′ UTR and/or a 3′ UTR may be provided asflanking regions. Multiple 5′ or 3′ UTRs may be included in the flankingregions and may be the same or of different sequences. Any portion ofthe flanking regions, including none, may be codon optimized and any mayindependently contain one or more different structural or chemicalmodifications, before and/or after codon optimization. Combinations offeatures may be included in the first and second flanking regions andmay be contained within other features. For example, the ORF may beflanked by a 5′ UTR which may contain a strong Kozak translationalinitiation signal and/or a 3′ UTR which may include an oligo(dT)sequence for templated addition of a poly-A tail.

Tables 2 and 3 provide a listing of exemplary UTRs which may be utilizedin the primary construct of the present invention as flanking regions.Shown in Table 2 is a representative listing of a 5′-untranslated regionof the invention. Variants of 5′ UTRs may be utilized wherein one ormore nucleotides are added or removed to the termini, including A, T, Cor G.

TABLE 2 5′-Untranslated Regions SEQ 5′ UTR Name/ ID IdentifierDescription Sequence NO. 5UTR-001 UpstreamGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAAT UTR ATAAGAGCCACC 1 5UTR-002 UpstreamGGGAGATCAGAGAGAAAAGAAGAGTAAGAAGAAAT 2 UTR ATAAGAGCCACC 5UTR-003 UpstreamGGAATAAAAGTCTCAACACAACATATACAAAACAA UTRACGAATCTCAAGCAATCAAGCATTCTACTTCTATTGC 3AGCAATTTAAATCATTTCTTTTAAAGCAAAAGCAATTTTCTGAAAATTTTCACCATTTACGAACGATAGCAAC 5UTR-004 UpstreamGGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAA 4 UTR AGCCACC

Shown in Table 3 is a representative listing of 3′-untranslated regionsof the invention. Variants of 3′ UTRs may be utilized wherein one ormore nucleotides are added or removed to the termini, including A, T, Cor G.

TABLE 3 3′-Untranslated Regions SEQ 3′ UTR Name/ ID IdentifierDescription Sequence NO. 3UTR-001 CreatineGCGCCTGCCCACCTGCCACCGACTGCTGGAACCCAG 5 KinaseCCAGTGGGAGGGCCTGGCCCACCAGAGTCCTGCTCCCTCACTCCTCGCCCCGCCCCCTGTCCCAGAGTCCCACCTGGGGGCTCTCTCCACCCTTCTCAGAGTTCCAGTTTCAACCAGAGTTCCAACCAATGGGCTCCATCCTCTGGATTCTGGCCAATGAAATATCTCCCTGGCAGGGTCCTCTTCTTTTCCCAGAGCTCCACCCCAACCAGGAGCTCTAGTTAATGGAGAGCTCCCAGCACACTCGGAGCTTGTGCTTTGTCTCCACGCAAAGCGATAAATAAAAGCATTGGTGGCCTTTGGTCTTTGAATAAAGCCTGAGTAGGAA GTCTAGA 3UTR-002 MyoglobinGCCCCTGCCGCTCCCACCCCCACCCATCTGGGCCCCG 6GGTTCAAGAGAGAGCGGGGTCTGATCTCGTGTAGCCATATAGAGTTTGCTTCTGAGTGTCTGCTTTGTTTAGTAGAGGTGGGCAGGAGGAGCTGAGGGGCTGGGGCTG GGGTGTTGAAGTTGGCTTTGCATGCCCAGCGATGCGCCTCCCTGTGGGATGTCATCACCCTGGGAACCGGGAGTGGCCCTTGGCTCACTGTGTTCTGCATGGTTTGGATCTGAATTAATTGTCCTTTCTTCTAAATCCCAACCGAACTTCTTCCAACCTCCAAACTGGCTGTAACCCCAAATCCAAGCCATTAACTACACCTGACAGTAGCAATTGTCTGATTAATCACTGGCCCCTTGAAGACAGCAGAATGTCCCTTTGCAATGAGGAGGAGATCTGGGCTGGGCGGGCCAGCTGGGGAAGCATTTGACTATCTGGAACTTGTGTGTGCCTCCTCAGGTATGGCAGTGACTCACCTGGTTTTAATAAAACAACCTGCAACATCTCATGGTCTTTGAAT AAAGCCTGAGTAGGAAGTCTAGA 3UTR-003α-actin ACACACTCCACCTCCAGCACGCGACTTCTCAGGACG 7ACGAATCTTCTCAATGGGGGGGCGGCTGAGCTCCAGCCACCCCGCAGTCACTTTCTTTGTAACAACTTCCGTTGCTGCCATCGTAAACTGACACAGTGTTTATAACGTGTACATACATTAACTTATTACCTCATTTTGTTATTTTTCGAAACAAAGCCCTGTGGAAGAAAATGGAAAACTTG AAGAAGCATTAAAGTCATTCTGTTAAGCTGCGTAAATGGTCTTTGAATAAAGCCTGAGTAGGAAGTCTAGA 3UTR-004 AlbuminCATCACATTTAAAAGCATCTCAGCCTACCATGAGAA 8TAAGAGAAAGAAAATGAAGATCAAAAGCTTATTCATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAATCTAATAGAGTGGTACAGCACTGTTATTTTTCAAAGATGTGTTGCTATCCTGAAAATTCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCTTATTCCACTTCGGTAGAGGATTTCTAGTTTCTTGTGGGCTAATTAAATAAATCATTAATACTCTTCTAATGGTCTTTGAATAAAGCCTGAGT AGGAAGTCTAGA 3UTR-005 α-globinGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCT 9TCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGC ATCTAGA 3UTR-006 G-CSFGCCAAGCCCTCCCCATCCCATGTATTTATCTCTATTT 10AATATTTATGTCTATTTAAGCCTCATATTTAAAGACAGGGAAGAGCAGAACGGAGCCCCAGGCCTCTGTGTCCTTCCCTGCATTTCTGAGTTTCATTCTCCTGCCTGTAGCAGTGAGAAAAAGCTCCTGTCCTCCCATCCCCTGGACTGGGAGGTAGATAGGTAAATACCAAGTATTTATTACTATGACTGCTCCCCAGCCCTGGCTCTGCAATGGGCACTGGGATGAGCCGCTGTGAGCCCCTGGTCCTGAGGGTCCCCACCTGGGACCCTTGAGAGTATCAGGTCTCCCACGTGGGAGACAAGAAATCCCTGTTTAATATTTAAACAGCAGTGTTCCCCATCTGGGTCCTTGCACCCCTCACTCTGGCCTCAGCCGACTGCACAGCGGCCCCTGCATCCCCTTGGCTGTGAGGCCCCTGGACAAGCAGAGGTGGCCAGAGCTGGGAGGCATGGCCCTGGGGTCCCACGAATTTGCTGGGGAATCTCGTTTTTCTTCTTAAGACTTTTGGGACATGGTTTGACTCCCGAACATCACCGACGCGTCTCCTGTTTTTCTGGGTGGCCTCGGGACACCTGCCCTGCCCCCACGAGGGTCAGGACTGTGACTCTTTTTAGGGCCAGGCAGGTGCCTGGACATTTGCCTTGCTGGACGG GGACTGGGGATGTGGGAGGGAGCAGACAGGAGGAATCATGTCAGGCCTGTGTGTGAAAGGAAGCTCCACTGTCACCCTCCACCTCTTCACCCCCCACTCACCAGTGTCCCCTCCACTGTCACATTGTAACTGAACTTCAGGATAATAAAGTGTTTGCCTCCATGGTCTTTGAATAAAGCCTG AGTAGGAAGGCGGCCGCTCGAGCATGCATCTAGA3UTR-007 Col1a2; ACTCAATCTAAATTAAAAAAGAAAGAAATTTGAAAA 11 collagen,AACTTTCTCTTTGCCATTTCTTCTTCTTCTTTTTTAAC type I, alphaTGAAAGCTGAATCCTTCCATTTCTTCTGCACATCTAC 2TTGCTTAAATTGTGGGCAAAAGAGAAAAAGAAGGATTGATCAGAGCATTGTGCAATACAGTTTCATTAACTCCTTCCCCCGCTCCCCCAAAAATTTGAATTTTTTTTTCAACACTCTTACACCTGTTATGGAAAATGTCAACCTTTGTAAGAAAACCAAAATAAAAATTGAAAAATAAAAACCATAAACATTTGCACCACTTGTGGCTTTTGAATATCTTCCACAGAGGGAAGTTTAAAACCCAAACTTCCAAAGGTTTAAACTACCTCAAAACACTTTCCCATGAGTGTGATCCACATTGTTAGGTGCTGACCTAGACAGAGATGAACTGAGGTCCTTGTTTTGTTTTGTTCATAATACAAAGGTGCTAATTAATAGTATTTCAGATACTTGAAGAATGTTGATGGTGCTAGAAGAATTTGAGAAGAAATACTCCTGTATTGAGTTGTATCGTGTGGTGTATTTTTTAAAAAATTTGATTTAGCATTCATATTTTCCATCTTATTCCCAATTAAAAGTATGCAGATTATTTGCCCAAATCTTCTTCAGATTCAGCATTTGTTCTTTGCCAGTCTCATTTTCATCTTCTTCCATGGTTCCACAGAAGCTTTGTTTCTTGGGCAAGCAGAAAAATTAAATTGTACCTATTTTGTATATGTGAGATGTTTAAATAAATTGTGAAAAAAATGAAATAAAGC ATGTTTGGTTTTCCAAAAGAACATAT 3UTR-008Col6a2; CGCCGCCGCCCGGGCCCCGCAGTCGAGGGTCGTGAG collagen,CCCACCCCGTCCATGGTGCTAAGCGGGCCCGGGTCC 12 type VI,CACACGGCCAGCACCGCTGCTCACTCGGACGACGCC alpha 2CTGGGCCTGCACCTCTCCAGCTCCTCCCACGGGGTCCCCGTAGCCCCGGCCCCCGCCCAGCCCCAGGTCTCCCCAGGCCCTCCGCAGGCTGCCCGGCCTCCCTCCCCCTGCAGCCATCCCAAGGCTCCTGACCTACCTGGCCCCTGAGCTCTGGAGCAAGCCCTGACCCAATAAAGGCTTTG AACCCAT 3UTR-009 RPN1;GGGGCTAGAGCCCTCTCCGCACAGCGTGGAGACGGG 13 ribophorin IGCAAGGAGGGGGGTTATTAGGATTGGTGGTTTTGTTTTGCTTTGTTTAAAGCCGTGGGAAAATGGCACAACTTTACCTCTGTGGGAGATGCAACACTGAGAGCCAAGGGGTGGGAGTTGGGATAATTTTTATATAAAAGAAGTTTTTCCACTTTGAATTGCTAAAAGTGGCATTTTTCCTATGTGCAGTCACTCCTCTCATTTCTAAAATAGGGACGTGGCCAGGCACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGCTCACGAGGTCAGGAGATCGAGACTATCCTGGCTAACACGGTAAAACCCTGTCTCTACTAAAAGTACAAAAAATTAGCTGGGCGTGGTGGTGGGCACCTGTAGTCCCAGCTACTCGGGAGG CTGAGGCAGGAGAAAGGCATGAATCCAAGAGGCAGAGCTTGCAGTGAGCTGAGATCACGCCATTGCACTCCAGCCTGGGCAACAGTGTTAAGACTCTGTCTCAAATATAAATAAATAAATAAATAAATAAATAAATAAATAAA AATAAAGCGAGATGTTGCCCTCAAA 3UTR-010LRP1; low GGCCCTGCCCCGTCGGACTGCCCCCAGAAAGCCTCC 14 densityTGCCCCCTGCCAGTGAAGTCCTTCAGTGAGCCCCTCC lipoproteinCCAGCCAGCCCTTCCCTGGCCCCGCCGGATGTATAA receptor-ATGTAAAAATGAAGGAATTACATTTTATATGTGAGC relatedGAGCAAGCCGGCAAGCGAGCACAGTATTATTTCTCC protein 1ATCCCCTCCCTGCCTGCTCCTTGGCACCCCCATGCTGCCTTCAGGGAGACAGGCAGGGAGGGCTTGGGGCTGCACCTCCTACCCTCCCACCAGAACGCACCCCACTGGGAGAGCTGGTGGTGCAGCCTTCCCCTCCCTGTATAAGACACTTTGCCAAGGCTCTCCCCTCTCGCCCCATCCCTGCTTGCCCGCTCCCACAGCTTCCTGAGGGCTAATTCTGGGAAGGGAGAGTTCTTTGCTGCCCCTGTCTGGAAG ACGTGGCTCTGGGTGAGGTAGGCGGGAAAGGATGGAGTGTTTTAGTTCTTGGGGGAGGCCACCCCAAACCCCAGCCCCAACTCCAGGGGCACCTATGAGATGGCCATGCTCAACCCCCCTCCCAGACAGGCCCTCCCTGTCTCCAGGGCCCCCACCGAGGTTCCCAGGGCTGGAGACTTCCTCTGGTAAACATTCCTCCAGCCTCCCCTCCCCTGGGGACGCCAAGGAGGTGGGCCACACCCAGGAAGGGAA AGCGGGCAGCCCCGTTTTGGGGACGTGAACGTTTTAATAATTTTTGCTGAATTCCTTTACAACTAAATAACAC AGATATTGTTATAAATAAAATTGT 3UTR-011Nnt1; ATATTAAGGATCAAGCTGTTAGCTAATAATGCCACC 15 cardiotrophin-TCTGCAGTTTTGGGAACAGGCAAATAAAGTATCAGT likeATACATGGTGATGTACATCTGTAGCAAAGCTCTTGG cytokineAGAAAATGAAGACTGAAGAAAGCAAAGCAAAAACT factor 1GTATAGAGAGATTTTTCAAAAGCAGTAATCCCTCAATTTTAAAAAAGGATTGAAAATTCTAAATGTCTTTCTGTGCATATTTTTTGTGTTAGGAATCAAAAGTATTTTATAAAAGGAGAAAGAACAGCCTCATTTTAGATGTAGTCCTGTTGGATTTTTTATGCCTCCTCAGTAACCAGAAATGTTTTAAAAAACTAAGTGTTTAGGATTTCAAGACAACATTATACATGGCTCTGAAATATCTGACACAATGTAAACATTGCAGGCACCTGCATTTTATGTTTTTTTTTTTCAACAAATGTGACTAATTTGAAACTTTTATGAACTTCTGAGCTGTCCCCTTGCAATTCAACCGCAGTTTGAATTAATCATATCAAATCAGTTTTAATTTTTTAAATTGTACTTCAGAGTCTATATTTCAAGGGCACATTTTCTCACTACTATTTTAATACATTAAAGGACTAAATAATCTTTCAGAGATGCTGGAAACAAATCATTTGCTTTATATGTTTCATTAGAATACCAATGAAACATACAACTTGAAAATTAGTAATAGTATTTTTGAAGATCCCATTTCTAATTGGAGATCTCTTTAATTTCGATCAACTTATAATGTGTAGTACTATATTAAGTGCACTTGAGTGGAATTCAACATTTGACTAATAAAATGAGTTCATCATGTTGGCAAGTGATGTGGCAATTATCTCTGGTGACAAAAGAGTAAAATCAAATATTTCTGCCTGTTACAAATATCAAGGAAGACCTGCTACTATGAAATAGATGACATTAATCTGTCTTCACTGTTTATAATACGGATGGATTTTTTTTCAAATCAGTGTGTGTTTTGAGGTCTTATGTAATTGATGACATTTGAGAGAAATGGTGGCTTTTTTTAGCTACCTCTTTGTTCATTTAAGCACCAGTAAAGATCATGTCTTTTTATAGAAGTGTAGATTTTCTTTGTGACTTTGCTATCGTGCCTAAAGCTCTAAATATAGGTGAATGTGTGATGAATACTCAGATTATTTGTCTCTCTATATAATTAGTTTGGTACTAAGTTTCTCAAAAAATTATTAACACATGAAAGACAATCTCTAAACCAGAAAAAGAAGTAGTACAAATTTTGTTACTGTAATGCTCGCGTTTAGTGAGTTTAAAACACACAGTATCTTTTGGTTTTATAATCAGTTTCTATTTTGCTGTGCCTGAGATTAAGATCTGTGTATGTGTGTGTGTGTGTGTGTGCGTTTGTGTGTTAAAGCAGAAAAGACTTTTTTAAAAGTTTTAAGTGATAAATGCAATTTGTTAATTGATCTTAGATCACTAGTAAACTCAGGGCTGAATTATACCATGTATATTCTATTAGAAGAAAGTAAACACCATCTTTATTCCTGCCCTTTTTCTTCTCTCAAAGTAGTTGTAGTTATATCTAGAAAGAAGCAATTTTGATTTCTTGAAAAGGTAGTTCCTGCACTCAGTTTAAACTAAAAATAATCATACTTGGATTTTATTTATTTTTGTCATAGTAAAAATTTTAATTTATATATATTTTTATTTAGTATTATCTTATTCTTTGCTATTTGCCAATCCTTTGTCATCAATTGTGTTAAATGAATTGAAAATTCATGCCCTGTTCATTTTATTTTACTTTATTGGTTAGGATATTTAAAGGATTTTTGTATATATAATTTCTTAAATTAATATTCCAAAAGGTTAGTGGACTTAGATTATAAATTATGGCAAAAATCTAAAAACAACAAAAATGATTTTTATACATTCTATTTCATTATTCCTCTTTTTCCAATAAGTCATACAATTGGTAGATATGACTTATTTATTTTTGTATTATTCACTATATCTTTATGATATTTAAGTATAAATAATTAAAAAAATTTATTGTACCTTATAGTCTGTCACCAAAAAAAAAAAATTATCTGTAGGTAGTGAAATGCTAATGTTGATTTGTCTTTAAGGGCTTGTTAACTATCCTTTATTTTCTCATTTGTCTTAAATTAGGAGTTTGTGTTTAAATTACTCATCTAAGCAAAAAATGTATATAAATCCCATTACTGGGTATATACCCAAAGGATTATAAATCATGCTGCTATAAAGACACATGCACACGTATGTTTATTGCAGCACTATTCACAATAGCAAAGACTTGGAACCAACCCAAATGTCCATCAATGATAGACTTGATTAAGAAAATGTGCACATATACACCATGGAATACTATGCAGCCATAAAAAAGGATGAGTTCATGTCCTTTGTAGGGACATGGATAAAGCTGGAAACCATCATTCTGAGCAAACTATTGCAAGGACAGAAAACCAAACACTGCATGTTCTCACTCATAGGTGGGAATTGAACAATGAGAACACTTGGACACAAGGTGGGGAACACCACACACCAGGGCCTGTC ATGGGGTGGGGGGAGTGGGGAGGGATAGCATTAGGAGATATACCTAATGTAAATGATGAGTTAATGGGTGCAGCACACCAACATGGCACATGTATACATATGTAGCAAACCTGCACGTTGTGCACATGTACCCTAGAACTTAA AGTATAATTAAAAAAAAAAAGAAAACAGAAGCTATTTATAAAGAAGTTATTTGCTGAAATAAATGTGATCTTTCCCATTAAAAAAATAAAGAAATTTTGGGGTAAAAAAACACAATATATTGTATTCTTGAAAAATTCTAAGAGAGTGGATGTGAAGTGTTCTCACCACAAAAGTGATAACTAATTGAGGTAATGCACATATTAATTAGAAAGATTTTGTCATTCCACAATGTATATATACTTAAAAATATGTTATACACAATAAATACATACATTAAAAAATAAGTAA ATGTA 3UTR-012 Col6a1;CCCACCCTGCACGCCGGCACCAAACCCTGTCCTCCC 16 collagen,ACCCCTCCCCACTCATCACTAAACAGAGTAAAATGT type VIGATGCGAATTTTCCCGACCAACCTGATTCGCTAGATT alpha 1TTTTTTAAGGAAAAGCTTGGAAAGCCAGGACACAACGCTGCTGCCTGCTTTGTGCAGGGTCCTCCGGGGCTCAGCCCTGAGTTGGCATCACCTGCGCAGGGCCCTCTGGGGCTCAGCCCTGAGCTAGTGTCACCTGCACAGGGCCCTCTGAGGCTCAGCCCTGAGCTGGCGTCACCTGTGCAGGGCCCTCTGGGGCTCAGCCCTGAGCTGGCCTCACCTGGGTTCCCCACCCCGGGCTCTCCTGCCCTGCCCTCCTGCCCGCCCTCCCTCCTGCCTGCGCAGCTCCTTCCCTAGGCACCTCTGTGCTGCATCCCACCAGCCTGAGCAAGACGCCCTCTCGGGGCCTGTGCCGCACTAGCCTCCCTCTCCTCTGTCCCCATAGCTGGTTTTTCCCACCAATCCTCACCTAACAGTTACTTTACAATTAAACTCAAAGCAAGCTCTTCTCCTCAGCTTGGGGCAGCCATTGGCCTCTGTCTCGTTTTGGGAAACCAAGGTCAGGAGGCCGTTGCAGACATAAATCTCGGCGACTCGGCCCCGTCTCCTGAGGGTCCTGCTGGTGACCGGCCTGGACCTTGGCCCTACAGCCCTGGAGGCCGCTGCTGACCAGCACTGACCCCGACCTCAGAGAGTACTCGCAGGGGCGCTGGCTGCACTCAAGACCCTCGAGATTAACGGTGCTAACCCCGTCTGCTCCTCCCTCCCGCAGAGACTGGGGCCTGGACTGGACATGAGAGCCCCTTGGTGCCACAGAGGGCTGTGTCTTACTAGAAACAACGCAAACCTCTCCTTCCTCAGAATAGTGATGTGTTCGACGTTTTATCAAAGGCCCCCTTTCTATGTTCATGTTAGTTTTGCTCCTTCTGTGTTTTTTTCTGAACCATATCCATGTTGCTGACTTTTCCAAATAA AGGTTTTCACTCCTCTC 3UTR-013 Calr;AGAGGCCTGCCTCCAGGGCTGGACTGAGGCCTGAGC 17 calreticulinGCTCCTGCCGCAGAGCTGGCCGCGCCAAATAATGTCTCTGTGAGACTCGAGAACTTTCATTTTTTTCCAGGCTGGTTCGGATTTGGGGTGGATTTTGGTTTTGTTCCCCTCCTCCACTCTCCCCCACCCCCTCCCCGCCCTTTTTTTTTTTTTTTTTTAAACTGGTATTTTATCTTTGATTCTCCTTCAGCCCTCACCCCTGGTTCTCATCTTTCTTGATCAACATCTTTTCTTGCCTCTGTCCCCTTCTCTCATCTCTTAGCTCCCCTCCAACCTGGGGGGCAGTGGTGTGGAGAAGCCACAGGCCTGAGATTTCATCTGCTCTCCTTCCTGGAGCCCAGAGGAGGGCAGCAGAAGGGGGTGGTGTCT CCAACCCCCCAGCACTGAGGAAGAACGGGGCTCTTCTCATTTCACCCCTCCCTTTCTCCCCTGCCCCCAGGACTGGGCCACTTCTGGGTGGGGCAGTGGGTCCCAGATTGGCTCACACTGAGAATGTAAGAACTACAAACAAAAT TTCTATTAAATTAAATTTTGTGTCTCC3UTR-014 Col1a1; CTCCCTCCATCCCAACCTGGCTCCCTCCCACCCAACC 18 collagen,AACTTTCCCCCCAACCCGGAAACAGACAAGCAACCC type I, alphaAAACTGAACCCCCTCAAAAGCCAAAAAATGGGAGA 1CAATTTCACATGGACTTTGGAAAATATTTTTTTCCTTTGCATTCATCTCTCAAACTTAGTTTTTATCTTTGACCAACCGAACATGACCAAAAACCAAAAGTGCATTCAAC CTTACCAAAAAAAAAAAAAAAAAAAGAATAAATAAATAACTTTTTAAAAAAGGAAGCTTGGTCCACTTGCTTGAAGACCCATGCGGGGGTAAGTCCCTTTCTGCCCGTTGGGCTTATGAAACCCCAATGCTGCCCTTTCTGCTCCTTTCTCCACACCCCCCTTGGGGCCTCCCCTCCACTCCTTCCCAAATCTGTCTCCCCAGAAGACACAGGAAACAATGTATTGTCTGCCCAGCAATCAAAGGCAATGCTCAAACACCCAAGTGGCCCCCACCCTCAGCCCGCTCCTGCCCGCCCAGCACCCCCAGGCCCTGGGGGACCTGGGGTTCTCAGACTGCCAAAGAAGCCTTGCCATCTGGCGCTCCCATGGCTCTTGCAACATCTCCCCTTCGTTTTTGAGGGGGTCATGCCGGGGGAGCCACCAGCCCCTCACTG GGTTCGGAGGAGAGTCAGGAAGGGCCACGACAAAGCAGAAACATCGGATTTGGGGAACGCGTGTCAATCCCTTGTGCCGCAGGGCTGGGCGGGAGAGACTGTTCTGTTCCTTGTGTAACTGTGTTGCTGAAAGACTACCTCGTTCTTGTCTTGATGTGTCACCGGGGCAACTGCCTGGGGGCGGGGATGGGGGCAGGGTGGAAGCGGCTCCCCATTTTATACCAAAGGTGCTACATCTATGTGATGGGTGGGGTGGGGAGGGAATCACTGGTGCTATAGAAATTGAGATGCCCCCCCAGGCCAGCAAATGTTCCTTTTTGTTCAAAGTCTATTTTTATTCCTTGATATTTTTCTTTTTTTTTTTTTTTTTTTGTGGATGGGGACTTGTGAATTTTTCTAAAGGTGCTATTTAACATGGGAGGAGAGCGTGTGCGGCTCCAGCCCAGCCCGCTGCTCACTTTCCACCCTCTCTCCACCTGCCTCTGGCTTCTCAGGCCTCTGCTCTCCGACCTCTCTCCTCTGAAACCCTCCTCCACAGCTGCAGCCCATCCTCCCGGCTCCCTCCTAGTCTGTCCTGCGCTCCTCTGTCCCCGGGTTTCAGAGACAACTTCCCAAAGCACAAAGCAGTTTTTCCCCCTAGGGGTGGGAGGAAGCAAAAGACTCTGTACCTATTTTGTATGTGTATAATAATTTGAGATGTTTTTAATTATTTTGATTGCTGGAATAAAGCATGTGGAAATGACCCAAACATAATCCGCAGTGGCCTCC TAATTTCCTTCTTTGGAGTTGGGGGAGGGGTGACATGGGGAAGGGGCTTTGGGGTGATGGGCTTGCCTTCCATTCCTGCCCTTTCCCTCCCCACTATTCTCTTCTAGATCCCTCCATAACCCCACTCCCCTTTCTCTCACCCTTCTTATACCGCAAACCTTTCTACTTCCTCTTTCATTTTCTATTCTTGCAATTTCCTTGCACCTTTTCCAAATCCTCTTCTCCCCTGCAATACCATACAGGCAATCCACGTGCACAACACACACACACACTCTTCACATCTGGGGTTGTCCAAACCTCATACCCACTCCCCTTCAAGCCCATCCACTCTCCACCCCCTGGATGCCCTGCACTTGGTGGCGGTGGGATGCTCATGGATACTGGGAGGGTGAGGGGAGTGGAACCCGTGAGGAGGACCTGGGGGCCTCTCCTTGAACTGACATGAAGGGTCATCTGGCCTCTGCTCCCTTCTCACCCACGCTGACCTCCTGCCGAAGGAGCAACGCAACAGGAGAGGGGTCTGCTGAGCCTGGCGAGGGTCTGGGAGGGACCAGGAGGAAGGCGTGCTCCCTGCTCGCTGTCCTG GCCCTGGGGGAGTGAGGGAGACAGACACCTGGGAGAGCTGTGGGGAAGGCACTCGCACCGTGCTCTTGGGAAGGAAGGAGACCTGGCCCTGCTCACCACGGACTGGGTGCCTCGACCTCCTGAATCCCCAGAACACAACCCCCCTGGGCTGGGGTGGTCTGGGGAACCATCGTGCCCCC GCCTCCCGCCTACTCCTTTTTAAGCTT3UTR-015 Plod1; TTGGCCAGGCCTGACCCTCTTGGACCTTTCTTCTTTG 19 procollagen-CCGACAACCACTGCCCAGCAGCCTCTGGGACCTCGG lysine, 2-GGTCCCAGGGAACCCAGTCCAGCCTCCTGGCTGTTG oxoglutarateACTTCCCATTGCTCTTGGAGCCACCAATCAAAGAGA 5-TTCAAAGAGATTCCTGCAGGCCAGAGGCGGAACACA dioxygenaseCCTTTATGGCTGGGGCTCTCCGTGGTGTTCTGGACCC 1AGCCCCTGGAGACACCATTCACTTTTACTGCTTTGTAGTGACTCGTGCTCTCCAACCTGTCTTCCTGAAAAACCAAGGCCCCCTTCCCCCACCTCTTCCATGGGGTGAGACTTGAGCAGAACAGGGGCTTCCCCAAGTTGCCCAGAAAGACTGTCTGGGTGAGAAGCCATGGCCAGAGCTTCTCCCAGGCACAGGTGTTGCACCAGGGACTTCTGCTTCAAGTTTTGGGGTAAAGACACCTGGATCAGACTCCAAGGGCTGCCCTGAGTCTGGGACTTCTGCCTCCATGGCTGGTCATGAGAGCAAACCGTAGTCCCCTGGAGACA GCGACTCCAGAGAACCTCTTGGGAGACAGAAGAGGCATCTGTGCACAGCTCGATCTTCTACTTGCCTGTGGGGAGGGGAGTGACAGGTCCACACACCACACTGGGTCACCCTGTCCTGGATGCCTCTGAAGAGAGGGACAGACCGTCAGAAACTGGAGAGTTTCTATTAAAGGTCATTTA AACCA 3UTR-016 Nucb1TCCTCCGGGACCCCAGCCCTCAGGATTCCTGATGCTC 20 nucleobindin 1CAAGGCGACTGATGGGCGCTGGATGAAGTGGCACAGTCAGCTTCCCTGGGGGCTGGTGTCATGTTGGGCTCCTGGGGCGGGGGCACGGCCTGGCATTTCACGCATTGCTGCCACCCCAGGTCCACCTGTCTCCACTTTCACAGCCTCCAAGTCTGTGGCTCTTCCCTTCTGTCCTCCGAGGGGCTTGCCTTCTCTCGTGTCCAGTGAGGTGCTCAGTGATCGGCTTAACTTAGAGAAGCCCGCCCCCTCCCCTTCTCCGTCTGTCCCAAGAGGGTCTGCTCTGAGCCTGCGTTCCTAGGTGGCTCGGCCTCAGCTGCCTGGGTTGTGGCCGCCCTAGCATCCTGTATGCCCACAGCTACTGGAATCCCCGCTGCTGCTCCGGGCCAAGCTTCTGGTTGATTAATGAGGGCATGGGGTGGTCCCTCAAGACCTTCCCCTACCTTTTGTGGAACCAGTGATGCCTCAAAGACAGTGTCCCCTCCACAGCTGGGTGCCAGGGGCAGGGGATCCTCAGTATAGCCGGTGAACCCTGATACCAGGAGCCTGGGCCTCCCTGAACCCCTGGCTTCCAGCCATCTCATCGCCAGCCTCCTCCTGGACCTCTTGGCCCCCAGCCCCTTCCCCACACAGCCCCAGAAGGGTCCCAGAGCTGACCCCACTCCAGGACCTAGGCCCAGCCCCTCAGCCTCATCTGGAGCCCCTGAAGACCAGTCCCACCCACCTTTCTGGCCTCATCTGACACTGCTCCGCATCCTGCTGTGTGTCCTGTTCCATGTTCCGGTTCCATCCAAATACACTTTCT GGAACAAA 3UTR-017 α-globinGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGG 21CCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC

It should be understood that those listed in the previous tables areexamples and that any UTR from any gene may be incorporated into therespective first or second flanking region of the primary construct.Furthermore, multiple wild-type UTRs of any known gene may be utilized.It is also within the scope of the present invention to provideartificial UTRs which are not variants of wild type genes. These UTRs orportions thereof may be placed in the same orientation as in thetranscript from which they were selected or may be altered inorientation or location. Hence a 5′ or 3′ UTR may be inverted,shortened, lengthened, made chimeric with one or more other 5′ UTRs or3′ UTRs. As used herein, the term “altered” as it relates to a UTRsequence, means that the UTR has been changed in some way in relation toa reference sequence. For example, a 3′ or 5′ UTR may be alteredrelative to a wild type or native UTR by the change in orientation orlocation as taught above or may be altered by the inclusion ofadditional nucleotides, deletion of nucleotides, swapping ortransposition of nucleotides. Any of these changes producing an“altered” UTR (whether 3′ or 5′) comprise a variant UTR.

In one embodiment, a double, triple or quadruple UTR such as a 5′ or 3′UTR may be used. As used herein, a “double” UTR is one in which twocopies of the same UTR are encoded either in series or substantially inseries. For example, a double beta-globin 3′ UTR may be used asdescribed in US Patent publication 20100129877, the contents of whichare incorporated herein by reference in its entirety.

It is also within the scope of the present invention to have patternedUTRs. As used herein “patterned UTRs” are those UTRs which reflect arepeating or alternating pattern, such as ABABAB or AABBAABBAABB orABCABCABC or variants thereof repeated once, twice, or more than 3times. In these patterns, each letter, A, B, or C represent a differentUTR at the nucleotide level.

In one embodiment, flanking regions are selected from a family oftranscripts whose proteins share a common function, structure, featureof property. For example, polypeptides of interest may belong to afamily of proteins which are expressed in a particular cell, tissue orat some time during development. The UTRs from any of these genes may beswapped for any other UTR of the same or different family of proteins tocreate a new chimeric primary transcript. As used herein, a “family ofproteins” is used in the broadest sense to refer to a group of two ormore polypeptides of interest which share at least one function,structure, feature, localization, origin, or expression pattern.

After optimization (if desired), the primary construct components arereconstituted and transformed into a vector such as, but not limited to,plasmids, viruses, cosmids, and artificial chromosomes. For example, theoptimized construct may be reconstituted and transformed into chemicallycompetent E. coli, yeast, neurospora, maize, drosophila, etc. where highcopy plasmid-like or chromosome structures occur by methods describedherein.

Stop Codons

In one embodiment, the primary constructs of the present invention mayinclude at least two stop codons before the 3′ untranslated region(UTR). The stop codon may be selected from TGA, TAA and TAG. In oneembodiment, the primary constructs of the present invention include thestop codon TGA and one additional stop codon. In a further embodimentthe addition stop codon may be TAA.

Vector Amplification

The vector containing the primary construct is then amplified and theplasmid isolated and purified using methods known in the art such as,but not limited to, a maxi prep using the Invitrogen PURELINK™ HiPureMaxiprep Kit (Carlsbad, Calif.).

Plasmid Linearization

The plasmid may then be linearized using methods known in the art suchas, but not limited to, the use of restriction enzymes and buffers. Thelinearization reaction may be purified using methods including, forexample Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.), andHPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen'sstandard PURELINK™ PCR Kit (Carlsbad, Calif.). The purification methodmay be modified depending on the size of the linearization reactionwhich was conducted. The linearized plasmid is then used to generatecDNA for in vitro transcription (IVT) reactions.

cDNA Template Synthesis

A cDNA template may be synthesized by having a linearized plasmidundergo polymerase chain reaction (PCR). Table 4 is a listing of primersand probes that may be usefully in the PCR reactions of the presentinvention. It should be understood that the listing is not exhaustiveand that primer-probe design for any amplification is within the skillof those in the art. Probes may also contain chemically modified basesto increase base-pairing fidelity to the target molecule andbase-pairing strength. Such modifications may include 5-methyl-cytidine,2,6-di-amino-purine, 2′-fluoro, phosphoro-thioate, or locked nucleicacids.

TABLE 4 Primers and Probes Primer/ Probe Hybridization SEQ ID IdentifierSequence (5′-3′) target NO. UFP TTGGACCCTCGTACAGAAGCTAA cDNA Template 22TACG URP T_(x160)CTTCCTACTCAGGCTTTATTC cDNA Template 23 AAAGACCA GBA1CCTTGACCTTCTGGAACTTC Acid 24 glucocerebrosidase GBA2CCAAGCACTGAAACGGATAT Acid 25 glucocerebrosidase LUC1GATGAAAAGTGCTCCAAGGA Luciferase 26 LUC2 AACCGTGATGAAAAGGTACC Luciferase27 LUC3 TCATGCAGATTGGAAAGGTC Luciferase 28 GCSF1 CTTCTTGGACTGTCCAGAGGG-CSF 29 GCSF2 GCAGTCCCTGATACAAGAAC G-CSF 30 GCSF3 GATTGAAGGTGGCTCGCTACG-CSF 31 *UFP is universal forward primer; URP is universal reverseprimer.

In one embodiment, the cDNA may be submitted for sequencing analysisbefore undergoing transcription.

mRNA Production

The process of mRNA or mmRNA production may include, but is not limitedto, in vitro transcription, cDNA template removal and RNA clean-up, andmRNA capping and/or tailing reactions. Each of these steps may beimproved by using the novel enzymes or enzyme variants designed asdescribed herein.

In Vitro Transcription

The cDNA produced in the previous step may be transcribed using an invitro transcription (IVT) system. The system typically comprises atranscription buffer, nucleotide triphosphates (NTPs), an RNaseinhibitor and a polymerase. The NTPs may be manufactured in house, maybe selected from a supplier, or may be synthesized as described herein.The NTPs may be selected from, but are not limited to, those describedherein including natural and unnatural (modified) NTPs. The polymerasemay be selected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to, the novelpolymerases able to incorporate modified NTPs as well as thosepolymerases described by Liu (Esvelt et al. (Nature (2011)472(7344):499-503 and U.S. Publication No. 20110177495) which recognizealternate promoters, Ellington (Chelliserrykattil and Ellington, NatureBiotechnology (2004) 22(9):1155-1160) describing a T7 RNA polymerasevariant to transcribe 2′-O-methyl RNA and Sousa (Padilla and Sousa,Nucleic Acids Research (2002) 30(24): e128) describing a T7 RNApolymerase double mutant; herein incorporated by reference in theirentireties.

Polymerases

Any number of polymerases or variants thereof may be used in thepreparation of the primary constructs of the present invention.

In one embodiment, the polymerases used in the present invention fusedwith a capping enzyme to form a chimeric enzyme. As used herein, theterm “chimeric enzyme” or “fusion enzyme” means an enzyme that is not anative enzyme that is found in nature. The term “chimeric enzyme” and“fusion enzyme” encompass monomeric enzymes but also oligomeric enzymes.The term “monomeric enzyme” relates to a single-unit enzyme thatconsists of only one peptide chain. The term “oligomeric enzyme” refersto a multi-unit enzyme that consists of at least two polypeptide chains,linked together covalently or noncovalently.

Cellular transcription is couple to 5′ capping and other mRNApost-transcriptional processing (such as, but not limited to, splicingand polyadenylation) in various systems. Using a chimeric enzymecomprising both a polymerase and a capping enzyme could allow for highefficiency 5′ capping of polynucleotides, primary constructs and/ormmRNA of the present invention. As a non-limiting example, a Vacciniacapping enzyme can form a binary complex with Vaccinia virus RNApolymerase. The interaction of the capping enzyme and the RNA polymerasecan facilitate the capping of the nascent mRNA chains when their 5′ endsare extruded from the RNA binding pocket on the elongating RNApolymerase. As another non-limiting example, a chimeric enzyme of acapping enzyme NP868R (mRNA capping enzyme of the Africian Swine FeverVirus) and a T7 RNA polymerase or a T3 RNA polymerase or a SP6 RNApolymerase can be formed with a peptide linker or a Leucine-zipperpeptide fused to each enzyme (see e.g., U.S. Patent Publication No.US20130042334 and International Patent Publication No WO2011128444, eachof which is herein incorporated by reference in its entirety). Inanother non-limiting example, a chimeric enzyme may be formed with aVaccinia capping enzyme (D1 and/or D12) and a T7 RNA polymerase asdescribed in U.S. Patent Publication No. US20130042334 and InternationalPatent Publication No WO2011128444, each of which is herein incorporatedby reference in its entirety, herein incorporated by reference in itsentirety.

In one embodiment, chimeric enzymes for use in the present invention canbe made by the methods described by Jais in U.S. Patent Publication No.US20130042334 and International Patent Publication No WO2011128444, eachof which is herein incorporated by reference in its entirety, hereinincorporated by reference in its entirety. As a non-limiting example,the capping enzyme and the RNA polymerase are cloned into mammalianexpression vectors and transfected into HEK293 cells.

In another embodiment, chimeric enzymes for use in the present inventioncan be made by constructing bacterial expression plasmids encoding achimeric enzyme of a capping enzyme and a RNA polymerase. As anon-limiting example, a chimeric enzyme which can catalyze both the mRNAsynthesis and the 5′ capping reaction is formed from a bacterialexpression plasmid encoding a chimeric enzyme of Vaccinia capping enzyme(D1 and/or D12) and T7 RNA polymerase. In another non-limiting example,the Vaccinia capping enzyme D12 and the T7 RNA polymerase can be encodedon the same expression plasmid and the D1 Vaccinia capping enzyme isencoded on a separate bacterial plasmid. The two plasmids are thenco-expressed so the recombinant proteins will be linked together by theprotein-protein interaction of D1 and D12 protein.

In one embodiment, the chimeric enzyme for use with the presentinvention can be made by constructing bacterial expression plasmidsencoding a chimeric enzyme of a capping enzyme and a DND polymerasemutated to synthesize RNA. The bacterial plasmids may be ones known inthe art and/or bacterial plasmids which have been modified to be able tosynthesize the chimeric enzyme.

In one embodiment, the chimeric enzyme further comprises a His tag whichmay be used for affinity purification.

The chimeric enzyme may be purified before it is used to synthesize andcap the polynucleotides, primary constructs and/or mmRNA of the presentinvention.

Polymerases: RNA Polymerases

In one aspect, the polymerases used in the present invention may be RNApolymerases. RNA polymerases may be modified by inserting or deletingamino acids of the RNA polymerase sequence. As a non-limiting example,the RNA polymerase may be modified to exhibit an increased ability toincorporate a 2′-modified nucleotide triphosphate compared to anunmodified RNA polymerase (see International Publication WO2008078180and U.S. Pat. No. 8,101,385; herein incorporated by reference in theirentireties).

Variants may be obtained by evolving an RNA polymerase, optimizing theRNA polymerase amino acid and/or nucleic acid sequence and/or by usingother methods known in the art. As a non-limiting example, T7 RNApolymerase variants may be evolved using the phage-assisted continuousdirected evolution (PACE) system set out by Esvelt et al. (Nature (2011)472(7344):499-503 and U.S. Publication No. 20110177495; hereinincorporated by reference in their entireties). PACE may achievecontinuous selection by linking the desired activity to the productionof infectious progeny phage containing the evolving gene.

In PACE systems, host cells comprising accessory plasmids andmutagenesis plasmids flow into lagoons where the host cells are infectedwith selection phages. As shown in FIG. 2, host cells continuously flowthrough a single lagoon, where they are infected with selection phage(SP) encoding library members. Functional library members of theputative polymerase enzyme variants can induce production of proteinIII, encoded by gene III, from the accessory plasmid (AP) and releaseprogeny capable of infecting new host cells, while non-functionallibrary members do not. Increased mutagenesis may be triggered throughinduction of the mutagenesis plasmid (MP). Host cells flow out thelagoon on average faster than they can replicate, confining theaccumulation of mutations to replicating phage.

In one embodiment, the clones of T7 RNA polymerase may encode at leastone mutation such as, but not limited to, lysine at position 93substituted for threonine (K93T), I4M, A7T, E63V, V64D, A65E, D66Y,T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H, F182L, L196F,G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M267I, G280C, H300R,D351A, A354S, E356D, L360P, A383V, Y385C, D388Y, S397R, M401T, N410S,K450R, P451T, G452V, E484A, H523L, H524N, G542V, E565K, K577E, K577M,N601S, S684Y, L699I, K713E, N748D, Q754R, E775K, A827V, D851N or L864F.

In one embodiment, the optimal evolutionary pressure is found when thepIII levels are above the minimal threshold required to prevent phagewashout but below the amount needed to maximize infectious phageproduction. The window may be shifted by varying copy number of theaccessory plasmid or by altering the ribosome-binding site sequence ofgene III.

In one embodiment, evolved T7 RNA polymerase may be purified and assayedby methods known in the art such as, but not limited to cell-based or invitro activity assays.

In one embodiment, PACE may be used to evolve T7 RNA polymerase variantswhich are capable of initiating or effecting transcription withchemically modified NTPs in a template-directed manner. As anon-limiting example, T7 RNA polymerase evolved to initiate transcriptswith an alanine and/or a cystine instead of a guanine may be used toproduce the mmRNA of the present invention. As another non-limitingexample, an RNA polymerase enzyme, such as, but not limited to, T7 RNApolymerase, may be evolved to be capable of incorporating chemicalmodifications into RNA during in vitro transcription. The chemicalmodification may be selected from, but not limited to, any chemicalmodification known in the art and/or described herein.

In one embodiment, PACE may be used to evolve RNA polymerase enzymes tohave a greater transcription efficiency through GC-rich regions ascompared to wild type RNA polymerase enzymes. In a further embodiment,the RNA polymerase is T7 RNA polymerase According to this embodiment,the template provided to the PACE system would be prepared havingincreased GC content or longer than native runs of guanosine orcytosine. The resulting translated protein would then be coupled to, andtrigger the expression of the accessory plasmid thereby ensuringselection of polymerases capable of the GC read through.

In one embodiment, PACE may be used to evolve RNA polymerase enzymes tohave a greater transcription efficiency to produce RNA as compared towild type RNA polymerase enzymes. In a further embodiment, the RNApolymerase is T7 RNA polymerase and/or the RNA is mRNA.

In one embodiment, PACE may be used to evolve RNA polymerase enzymeswhich may be capable of in vitro transcription at a pH less than 3, lessthan 7 and/or greater than 7. Accordingly, the pH of the lagoon may bemodulated and the selection would proceed by using a standard n-hybridselection schema.

In one embodiment, the enzyme variants may be screened using methodsknown in the art to determine the enzyme variants which are capable oftranscribing mRNAs longer than wild type T7 RNA polymerase, capable oftranscribing through GC-rich regions, capable of incorporating aselection of modified nucleoside triphosphates in a transcript, capableof increasing the yield of transcripts and are able to function in invitro transcription in altered pH ranges.

In one embodiment, the synthesis of RNA may be catalyzed by contacting aDNA template with a T7 RNA polymerase variant which is capable ofincorporating chemical modification into RNA during in vitrotranscription, in the presence of chemically modified nucleosidetriphosphates (NTPs). In a further embodiment, the chemically modifiedNTPs differ from cytidine, adenosine, guanosine and uridine by amodification to the ribofuranosyl ring. In another embodiment, thechemically modified NTPs comprise at least one chemical modificationsuch as, but not limited to, thoses known in the art and/or describedherein. The NTP may be fully modified, at least 50% modified or lessthan 50% modified.

In one embodiment, RNA polymerase described herein for use with RNA suchas, but not limited to, modified mRNA, may be a RNA polymerase from avirus of the Caliciviridae family. RNA polymerases of viruses of theCaliciviridae family are known to polymerize a complementary RNA strandon an RNA template in the presence or absence of a primer (see e.g,W0200712329 and WO2012038450, the contents of each of which are hereinincorporated by reference in their entirety). RNA polymerases of theviruses of the Caliciviridae family have a “right hand conformation” andthe amino acid sequences of the polymerases comprise a conservedarrangement as described in WO2012038450, the contents of which areherein incorporated by reference in its entirety. The “right handconformation” means that the tertiary structure (conformation) of theRNA polymerase folds like a right hand with finger, palm and thumb. Thisis a common conformation in most template-dependent polymerases.

In one embodiment, isolated mRNA and/or in vitro synthesized mRNA may bepost-transcriptionally modified. The mRNA may be contacted with anenzyme variant known in the art, described herein and/or produced usingphage-assisted continuous evolution. In one embodiment, thepost-transcriptional modification is selected from, but not limited to,methylation and pseduouridylation. In one embodiment, the mRNApost-transcriptional modification is methylation and the enzyme variantis methyl transferase. In another embodiment, the mRNApost-transcriptional modification is pseudouridylation and the enzymevariant is pseudouridine synthase.

As another non-limiting example, T7 RNA polymerase variants may encodeat least mutation as described in U.S. Pub. Nos. 20100120024 and20070117112; herein incorporated by reference in their entireties.Variants of RNA polymerase may also include, but are not limited to,substitutional variants, conservative amino acid substitution,insertional variants, deletional variants and/or covalent derivatives.

In one embodiment, the primary construct may be designed to berecognized by the wild type or variant RNA polymerases. In doing so, theprimary construct may be modified to contain sites or regions ofsequence changes from the wild type or parent primary construct.

In one embodiment, the primary construct may be designed to include atleast one substitution and/or insertion upstream of an RNA polymerasebinding or recognition site, downstream of the RNA polymerase binding orrecognition site, upstream of the TATA box sequence, downstream of theTATA box sequence of the primary construct but upstream of the codingregion of the primary construct, within the 5′UTR, before the 5′UTRand/or after the 5′UTR.

In one embodiment, the 5′UTR of the primary construct may be replaced bythe insertion of at least one region and/or string of nucleotides of thesame base. The region and/or string of nucleotides may include, but isnot limited to, at least 3, at least 4, at least 5, at least 6, at least7 or at least 8 nucleotides and the nucleotides may be natural and/orunnatural. As a non-limiting example, the group of nucleotides mayinclude 5-8 adenine, cytosine, thymine, a string of any of the othernucleotides disclosed herein and/or combinations thereof.

In one embodiment, the 5′UTR of the primary construct may be replaced bythe insertion of at least two regions and/or strings of nucleotides oftwo different bases such as, but not limited to, adenine, cytosine,thymine, any of the other nucleotides disclosed herein and/orcombinations thereof. For example, the 5′UTR may be replaced byinserting 5-8 adenine bases followed by the insertion of 5-8 cytosinebases. In another example, the 5′UTR may be replaced by inserting 5-8cytosine bases followed by the insertion of 5-8 adenine bases.

In one embodiment, the primary construct may include at least onesubstitution and/or insertion downstream of the transcription start sitewhich may be recognized by an RNA polymerase. As a non-limiting example,at least one substitution and/or insertion may occur downstream thetranscription start site by substituting at least one nucleic acid inthe region just downstream of the transcription start site (such as, butnot limited to, +1 to +6). Changes to region of nucleotides justdownstream of the transcription start site may affect initiation rates,increase apparent nucleotide triphosphate (NTP) reaction constantvalues, and increase the dissociation of short transcripts from thetranscription complex curing initial transcription (Brieba et al,Biochemistry (2002) 41: 5144-5149; herein incorporated by reference inits entirety). The modification, substitution and/or insertion of atleast one nucleic acid may cause a silent mutation of the nucleic acidsequence or may cause a mutation in the amino acid sequence.

In one embodiment, the primary construct may include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12 or at least 13 guanine bases downstream of the transcription startsite.

In one embodiment, the primary construct may include the substitution ofat least 1, at least 2, at least 3, at least 4, at least 5 or at least 6guanine bases in the region just downstream of the transcription startsite. As a non-limiting example, if the nucleotides in the region areGGGAGA the guanine bases may be substituted by at least 1, at least 2,at least 3 or at least 4 adenine nucleotides. In another non-limitingexample, if the nucleotides in the region are GGGAGA the guanine basesmay be substituted by at least 1, at least 2, at least 3 or at least 4cytosine bases. In another non-limiting example, if the nucleotides inthe region are GGGAGA the guanine bases may be substituted by at least1, at least 2, at least 3 or at least 4 thymine, and/or any of thenucleotides described herein.

In one embodiment, the primary construct may include at least onesubstitution and/or insertion upstream of the start codon. For thepurpose of clarity, one of skill in the art would appreciate that thestart codon is the first codon of the protein coding region whereas thetranscription start site is the site where transcription begins. Theprimary construct may include, but is not limited to, at least 1, atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7 orat least 8 substitutions and/or insertions of nucleotide bases. Thenucleotide bases may be inserted or substituted at 1, at least 1, atleast 2, at least 3, at least 4 or at least 5 locations upstream of thestart codon. The nucleotides inserted and/or substituted may be the samebase (e.g., all A or all C or all T or all G), two different bases(e.g., A and C, A and T, or C and T), three different bases (e.g., A, Cand T or A, C and T) or at least four different bases. As a non-limitingexample, the guanine base upstream of the coding region in the primaryconstruct may be substituted with adenine, cytosine, thymine, or any ofthe nucleotides described herein. In another non-limiting example thesubstitution of guanine bases in the primary construct may be designedso as to leave one guanine base in the region downstream of thetranscription start site and before the start codon (see Esvelt et al.Nature (2011) 472(7344):499-503; herein incorporated by reference in itsentirety). As a non-limiting example, at least 5 nucleotides may beinserted at 1 location downstream of the transcription start site butupstream of the start codon and the at least 5 nucleotides may be thesame base type.

Polymerases: DNA Polymerases

In another aspect, the polymerases used in the present invention may beDNA polymerases that can synthesize RNAs. The DNA polymerases maycomprise one or more mutations allowing the DNA polymerase to make RNA(see e.g., International Patent Publication No. WO2011135280, U.S.Patent Application No US20130130320 and Cozens et al. A short adaptivepath from DNA to RNA polymerases. PNAS 2012:109(21) 8067-8072, each ofwhich is herein incorporated by reference in its entirety). In oneaspect, the DNA polymerase may comprise backbone modifications and/ormay be truncated in order to produce RNA at the desired efficiency rate.In another aspect, the DNA polymerase may have a polymerase backbonefrom the polB enzyme family (see e.g., International Patent PublicationNo. WO2011135280 and U.S. Patent Application No US20130130320, each ofwhich is herein incorporated by reference in its entirety). Mutations ofDNA polymerase backbones for polB enzyme may be transferred to otherB-family polymerases. As a non-limiting example, the mutations of theDNA polymerase having the polymerase backbone Archaeal thermococcusTgoTpolB (Tgo (1TGO)) may be transferred to other B-family polymerasessuch as, but not limited to, Pol6G12, RB69 (1IG9) or E. coli (3MAQ) asdescribed in International Patent Publication No. WO2011135280 and U.S.Patent Application No US20130130320, each of which is hereinincorporated by reference in its entirety, and shown below in Table 5.In Table 5, residues that were not mapped to equivalent positions inInternational Patent Publication No. WO2011135280 or U.S. PatentApplication No US20130130320 are shown as N.D.

TABLE 5 Mapping of DNA Polymerase Backbone Mutations Tgo (1TGO) PoI6G12RB69 E. coli Amino Position Amino Acid (1IG9) (3MAQ) acid No. ChangeResidue No. Residue No. V 589 A 703 604 E 609 K 732 N.D. I 610 M 733N.D. K 659 Q 778 681 E 664 Q 783 686 Q 665 P 784 687 R 668 K 788 690 D669 Q 789 691 K 671 H N.D 693 K 674 R 792 N.D. T 676 R 801 700 A 681 S806 705 L 704 P 835 733 E 730 G 869 750

As a non-limiting example, the DNA polymerase may have the polymerasebackbone Archaeal thermococcus TgoTpolB (see e.g., International PatentPublication No. WO2011135280, U.S. Patent Application No US20130130320and Cozens et al. A short adaptive path from DNA to RNA polymerases.PNAS 2012:109(21) 8067-8072, each of which is herein incorporated byreference in its entirety). The DNA polymerase having the polymerasebackbone Archaeal thermococcus TgoTpolB may have the wild-type aminoacid sequence shown in SEQ ID NO: 32, a fragment thereof or a sequencethat is at least 99% identical, at least 95% identical, at least 90%identical, at least 85% identical, at least 80% identical, at least 75%identical, at least 70% identical, at least 65% identical, at least 60%identical, at least 55% identical, at least 50% identical, at least 45%identical, at least 40% identical or at least 35% identical. As anon-limiting example, the DNA polymerase which has been mutated to beable to synthesize mRNA has the amino acid sequence shown in SEQ ID NO:2-18, 37-39, 41, 43-45 of U. S. Patent Application No US20130130320(herein incorporated by reference in its entirety) or the DNA sequenceshown in SEQ ID NO: 19-36, 40 or 42 of U. S. Patent Application NoUS20130130320 (herein incorporated by reference in its entirety), afragment or a variant thereof that may have at least 99% identity, atleast 95% identity, at least 90% identity, at least 85% identity, atleast 80% identity, at least 75% identity, at least 70% identity, atleast 65% identity, at least 60% identity, at least 55% identity, atleast 50% identity, at least 45% identity, at least 40% identity or atleast 35% identity. In another aspect, the DNA polymerase having thepolymerase backbone Archaeal thermococcus TgoTpolB may have one or moremutations in the region of amino acid 651 to amino acid 679. The regionof amino acid 662 to amino acid 666 may comprise a mutation, themutation may be located, for example, at position 664 (e.g., E664Q orE664K as described in International Patent Publication No. WO2011135280and U.S. Patent Application No US20130130320, each of which is hereinincorporated by reference in its entirety).

As another non-limiting example, the DNA polymerase may be derived froma variant of Tgo, the hyperthermophilic archaeon Thermococcusgorgonarius, containing mutations to disable read-ahead stalling (V93Q)and the exonuclease domain (D141A, E143A) and the Therminator mutation(A485L) which enhances the incorporation of unnatural substances. TheDNA polymerase may be the polymerase known as “D4” described inInternational Patent Publication No. WO2011135280, U.S. PatentApplication No US20130130320 and Cozens et al. A short adaptive pathfrom DNA to RNA polymerases. PNAS 2012:109(21) 8067-8072, each of whichis herein incorporated by reference in its entirety. D4 contains themutations of the variant Tgo and nine additional mutations comprising acluster of eight mutations (P657T, E658Q, K659H, Y663H, E664Q, D669A,K671N and T676I) in the thumb sub-domain which is residues 586-773 and asingle mutation (L403P) in the A-motif (see e.g., International PatentPublication No. WO2011135280, U.S. Patent Application No US20130130320and Cozens et al. A short adaptive path from DNA to RNA polymerases.PNAS 2012:109(21) 8067-8072, each of which is herein incorporated byreference in its entirety).

In yet another non-limiting example, the DNA polymerase may comprise atleast two mutations. The DNA polymerase may be Tgo or a variant thereofand may comprise a mutation at the amino acid 664 (e.g., E664K) and mayalso comprise a mutation at amino acid position 409 (e.g., Y409G) (seee.g., International Patent Publication No. WO2011135280, U.S. PatentApplication No US20130130320 and Cozens et al. A short adaptive pathfrom DNA to RNA polymerases. PNAS 2012:109(21) 8067-8072, each of whichis herein incorporated by reference in its entirety).

cDNA Template Removal and Clean-Up

The cDNA template may be removed using methods known in the art such as,but not limited to, treatment with Deoxyribonuclease I (DNase I). RNAclean-up may also include a purification method such as, but not limitedto, AGENCOURT® CLEANSEQ® system from Beckman Coulter (Danvers, Mass.),HPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

Capping and/or Tailing Reactions

The primary construct or mmRNA may also undergo capping and/or tailingreactions. A capping reaction may be performed by methods known in theart to add a 5′ cap to the 5′ end of the primary construct. Methods forcapping include, but are not limited to, using a Vaccinia Capping enzyme(New England Biolabs, Ipswich, Mass.).

A poly-A tailing reaction may be performed by methods known in the art,such as, but not limited to, 2′ O-methyltransferase and by methods asdescribed herein. If the primary construct generated from cDNA does notinclude a poly-T, it may be beneficial to perform the poly-A-tailingreaction before the primary construct is cleaned.

mRNA Purification

Primary construct or mmRNA purification may include, but is not limitedto, mRNA or mmRNA clean-up, quality assurance and quality control. mRNAor mmRNA clean-up may be performed by methods known in the arts such as,but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers,Mass.), poly-T beads, LNA™ oligo-T capture probes (EXIQON® Inc, Vedbaek,Denmark) or HPLC based purification methods such as, but not limited to,strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term“purified” when used in relation to a polynucleotide such as a “purifiedmRNA or mmRNA” refers to one that is separated from at least onecontaminant. As used herein, a “contaminant” is any substance whichmakes another unfit, impure or inferior. Thus, a purified polynucleotide(e.g., DNA and RNA) is present in a form or setting different from thatin which it is found in nature, or a form or setting different from thatwhich existed prior to subjecting it to a treatment or purificationmethod.

A quality assurance and/or quality control check may be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC.

In another embodiment, the mRNA or mmRNA may be sequenced by methodsincluding, but not limited to reverse-transcriptase-PCR.

In one embodiment, the mRNA or mmRNA may be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, Mass.). The quantified mRNA ormmRNA may be analyzed in order to determine if the mRNA or mmRNA may beof proper size, check that no degradation of the mRNA or mmRNA hasoccurred. Degradation of the mRNA and/or mmRNA may be checked by methodssuch as, but not limited to, agarose gel electrophoresis, HPLC basedpurification methods such as, but not limited to, strong anion exchangeHPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), andhydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-massspectrometry (LCMS), capillary electrophoresis (CE) and capillary gelelectrophoresis (CGE).

Signal Sequences

The primary constructs or mmRNA may also encode additional featureswhich facilitate trafficking of the polypeptides to therapeuticallyrelevant sites. One such feature which aids in protein trafficking isthe signal sequence. As used herein, a “signal sequence” or “signalpeptide” is a polynucleotide or polypeptide, respectively, which is fromabout 9 to 200 nucleotides (3-60 amino acids) in length which isincorporated at the 5′ (or N-terminus) of the coding region orpolypeptide encoded, respectively. Addition of these sequences result intrafficking of the encoded polypeptide to the endoplasmic reticulumthrough one or more secretory pathways. Some signal peptides are cleavedfrom the protein by signal peptidase after the proteins are transported.

Table 6 is a representative listing of protein signal sequences whichmay be incorporated for encoding by the polynucleotides, primaryconstructs or mmRNA of the invention.

TABLE 6 Signal Sequences NUCLEOTIDE SEQUENCE SEQ ID ENCODED SEQ ID IDDescription (5′-3′) NO. PEPTIDE NO. SS- α-1- ATGATGCCATCCTCAGTCT 33MMPSSVSWGILL 95 001 antitrypsin CATGGGGTATTTTGCTCTTG AGLCCLVPVSLAGCGGGTCTGTGCTGTCTCG TGCCGGTGTCGCTCGCA SS- G-CSF ATGGCCGGACCGGCGACTC 34MAGPATQSPMKL 96 002 AGTCGCCCATGAAACTCAT MALQLLLWHSA GGCCCTGCAGTTGTTGCTTTLWTVQEA GGCACTCAGCCCTCTGGAC CGTCCAAGAGGCG SS- Factor IXATGCAGAGAGTGAACATGA 35 MQRVNMIMAESP 97 003 TTATGGCCGAGTCCCCATCSLITICLLGYLLSA GCTCATCACAATCTGCCTG ECTVFLDHENAN CTTGGTACCTGCTTTCCGCCKILNRPKR GAATGCACTGTCTTTCTGG ATCACGAGAATGCGAATAA GATCTTGAACCGACCCAAA CGGSS- Prolactin ATGAAAGGATCATTGCTGT 36 MKGSLLLLLVSN 98 004TGCTCCTCGTGTCGAACCTT LLLCQSVAP CTGCTTTGCCAGTCCGTAG CCCCC SS- AlbuminATGAAATGGGTGACGTTCA 37 MKWVTFISLLFLF 99 005 TCTCACTGTTGTTTTTGTTC SSAYSRGVFRR TCGTCCGCCTACTCCAGGG GAGTATTCCGCCGA SS- HMMSP38 ATGTGGTGGCGGCTCTGGT8 MWWRLWWLLL 100 006 GGCTGCTCCTGTTGCTCCTC LLLLLPMWA TTGCTGTGGCCCATGGTGTGGGCA MLS- ornithine TGCTCTTTAACCTCCGCATC 39 MLFNLRILLNNA 101 001carbamoyltransferase CTGTTGAATAACGCTGCGT AFRNGHNFMVR TCCGAAATGGGCATAACTTNFRCGQPLQ CATGGTACGCAACTTCAGA TGCGGCCAGCCACTCCAG MLS- CytochromeATGTCCGTCTTGACACCCC 40 MSVLTPLLLRGL 102 002 C OxidaseTGCTCTTGAGAGGGCTGAC TGSARRLPVPRA subunit 8A GGGGTCCGCTAGACGCCTG KIHSLCCGGTACCGCGAGCGAAGA TCCACTCCCTG MLS- Cytochrome ATGAGCGTGCTCACTCCGT 41MSVLTPLLLRGL 103 003 C Oxidase TGCTTCTTCGAGGGCTTAC TGSARRLPVPRA subunit8A GGGATCGGCTCGGAGGTTG KIHSL CCCGTCCCGAGAGCGAAGA TCCATTCGTTG SS- TypeIII, TGACAAAAATAACTTTATC 42 MVTKITLSPQNFR 104 007 bacterialTCCCCAGAATTTTAGAATC IQKQETTLLKEKS CAAAAACAGGAAACCACA TEKNSLAKSILAVCTACTAAAAGAAAAATCAA KNHFIELRSKLSE CCGAGAAAAATTCTTTAGC RFISHKNTAAAAAGTATTCTCGCAGTA AAAATCACTTCATCGAATT AAGGTCAAAATTATCGGAACGTTTTATTTCGCATAAGA ACACT SS- Viral ATGCTGAGCTTTGTGGATA 43 MLSFVDTRTLLL105 008 CCCGCACCCTGCTGCTGCT LAVTSCLATCQ GGCGGTGACCAGCTGCCTG GCGACCTGCCAGSS- viral ATGGGCAGCAGCCAGGCGC 44 MGSSQAPRMGS 106 009 CGCGCATGGGCAGCGTGGGVGGHGLMALLM CGGCCATGGCCTGATGGCG AGLILPGILA CTGCTGATGGCGGGCCTGATTCTGCCGGGCATTCTGGCG SS- Viral ATGGCGGGCATTTTTTATTT 45 MAGIFYFLFSFLF 107010 TCTGTTTAGCTTTCTGTTTG GICD GCATTTGCGAT SS- Viral ATGGAAAACCGCCTGCTGC46 MENRLLRVFLV 108 011 GCGTGTTTCTGGTGTGGGC WAALTMDGASAGGCGCTGACCATGGATGGC GCGAGCGCG SS- Viral ATGGCGCGCCAGGGCTGCT 47MARQGCFGSYQ 109 012 TTGGCAGCTATCAGGTGAT VISLFTFAIGVNLTAGCCTGTTTACCTTTGCGA CLG TTGGCGTGAACCTGTGCCT GGGC SS- BacillusATGAGCCGCCTGCCGGTGC 48 MSRLPVLLLLQL 110 013 TGCTGCTGCTGCAGCTGCT LVRPGLQGGTGCGCCCGGGCCTGCAG SS- Bacillus ATGAAACAGCAGAAACGC 49 MKQQKRLYARL 111014 CTGTATGCGCGCCTGCTGA LTLLFALIFLLPHS CCCTGCTGTTTGCGCTGATT SASATTTCTGCTGCCGCATAGCA GCGCGAGCGCG SS- Secretion ATGGCGACGCCGCTGCCTC 50MATPLPPPSPRHL 112 015 signal CGCCCTCCCCGCGGCACCT RLLRLLLSGGCGGCTGCTGCGGCTGCTG CTCTCCGCCCTCGTCCTCGGC SS- SecretionATGAAGGCTCCGGGTCGGC 51 MKAPGRLVLIILC 113 016 signal TCGTGCTCATCATCCTGTGCSVVFS TCCGTGGTCTTCTCT SS- Secretion ATGCTTCAGCTTTGGAAAC 52 MLQLWKLLCGV114 017 signal TTGTTCTCCTGTGCGGCGTG LT CTCACT SS- SecretionATGCTTTATCTCCAGGGTT 53 MLYLQGWSMPA 115 018 signal GGAGCATGCCTGCTGTGGCAVA SS- Secretion ATGGATAACGTGCAGCCGA 54 MDNVQPKIKHRP 116 019 signalAAATAAAACATCGCCCCTT FCFSVKGHVKML CTGCTTCAGTGTGAAAGGC RLDIINSLVTTVFCACGTGAAGATGCTGCGGC MLIVSVLALIP TGGATATTATCAACTCACT GGTAACAACAGTATTCATGCTCATCGTATCTGTGTTGGC ACTGATACCA SS- Secretion ATGCCCTGCCTAGACCAAC 55MPCLDQQLTVHA 117 020 signal AGCTCACTGTTCATGCCCT LPCPAQPSSLAFCACCCTGCCCTGCCCAGCCC QVGFLTA TCCTCTCTGGCCTTCTGCCA AGTGGGGTTCTTAACAGCA SS-Secretion ATGAAAACCTTGTTCAATC 56 MKTLFNPAPAIA 118 021 signalCAGCCCCTGCCATTGCTGA DLDPQFYTLSDV CCTGGATCCCCAGTTCTAC FCCNESEAEILTGACCCTCTCAGATGTGTTCT LTVGSAADA GCTGCAATGAAAGTGAGGC TGAGATTTTAACTGGCCTCACGGTGGGCAGCGCTGCAG ATGCT SS- Secretion ATGAAGCCTCTCCTTGTTGT 57MKPLLVVFVFLF 119 022 signal GTTTGTCTTTCTTTTCCTTT LWDPVLAGGGATCCAGTGCTGGCA SS- Secretion ATGTCCTGTTCCCTAAAGTT 58 MSCSLKFTLIVIFF120 023 signal TACTTTGATTGTAATTTTTT TCTLSSS TTTACTGTTGGCTTTCATCC AGC SS-Secretion ATGGTTCTTACTAAACCTCT 59 MVLTKPLQRNGS 121 024 signalTCAAAGAAATGGCAGCATG MMSFENVKEKSR ATGAGCTTTGAAAATGTGA EGGPHAHTPEEEAAGAAAAGAGCAGAGAAG LCFVVTHTPQVQ GAGGGCCCCATGCACACAC TTLNLFFHIFKVLACCCGAAGAAGAATTGTGT TQPLSLLWG TTCGTGGTAACACACTACC CTCAGGTTCAGACCACACTCAACCTGTTTTTCCATATAT TCAAGGTTCTTACTCAACC ACTTTCCCTTCTGTGGGGT SS-Secretion ATGGCCACCCCGCCATTCC 60 MATPPFRLIRKM 122 025 signalGGCTGATAAGGAAGATGTT FSFKVSRWMGLA TTCCTTCAAGGTGAGCAGA CFRSLAASTGGATGGGGCTTGCCTGCT TCCGGTCCCTGGCGGCATCC SS- SecretionATGAGCTTTTTCCAACTCCT 61 MSFFQLLMKRKE 123 026 signal GATGAAAAGGAAGGAACTLIPLVVFMTVAA CATTCCCTTGGTGGTGTTCA GGASS TGACTGTGGCGGCGGGTGG AGCCTCATCTSS- Secretion ATGGTCTCAGCTCTGCGGG 62 MVSALRGAPLIR 124 027 signalGAGCACCCCTGATCAGGGT VHSSPVSSPSVSG GCACTCAAGCCCTGTTTCTT PAALVSCLSSQSSCTCCTTCTGTGAGTGGACC ALS ACGGAGGCTGGTGAGCTGC CTGTCATCCCAAAGCTCAG CTCTGAGCSS- Secretion ATGATGGGGTCCCCAGTGA 63 MMGSPVSHLLAG 125 028 signalGTCATCTGCTGGCCGGCTT FCVWVVLG CTGTGTGTGGGTCGTCTTG GGC SS- SecretionATGGCAAGCATGGCTGCCG 64 MASMAAVLTWA 126 029 signal TGCTCACCTGGGCTCTGGCLALLSAFSATQA TCTTCTTTCAGCGTTTTCGG CCACCCAGGCA SS- SecretionATGGTGCTCATGTGGACCA 65 MVLMWTSGDAF 127 030 signal GTGGTGACGCCTTCAAGACKTAYFLLKGAPL GGCCTACTTCCTGCTGAAG QFSVCGLLQVLV GGTGCCCCTCTGCAGTTCTDLAILGQATA CCGTGTGCGGCCTGCTGCA GGTGCTGGTGGACCTGGCC ATCCTGGGGCAGGCCTACGCC SS- Secretion ATGGATTTTGTCGCTGGAG 66 MDFVAGAIGGVC 128 031 signalCCATCGGAGGCGTCTGCGG GVAVGYPLDTVK TGTTGCTGTGGGCTACCCC VRIQTEPLYTGIWCTGGACACGGTGAAGGTCA HCVRDTYHRERV GGATCCAGACGGAGCCAA WGFYRGLSLPVCAGTACACAGGCATCTGGCA TVSLVSS CTGCGTCCGGGATACGTAT CACCGAGAGCGCGTGTGGGGCTTCTACCGGGGCCTCTC GCTGCCCGTGTGCACGGTG TCCCTGGTATCTTCC SS- SecretionATGGAGAAGCCCCTCTTCC 67 MEKPLFPLVPLH 129 032 signal CATTAGTGCCTTTGCATTGWFGFGYTALVVS GTTTGGCTTTGGCTACACA GGIVGYVKTGSV GCACTGGTTGTTTCTGGTGPSLAAGLLFGSLA GGATCGTTGGCTATGTAAA AACAGGCAGCGTGCCGTCCCTGGCTGCAGGGCTGCTCT TCGGCAGTCTAGCC SS- Secretion ATGGGTCTGCTCCTTCCCCT 68MGLLLPLALCILV 130 033 signal GGCACTCTGCATCCTAGTC LC CTGTGC SS- SecretionATGGGGATCCAGACGAGCC 69 MGIQTSPVLLASL 131 034 signal CCGTCCTGCTGGCCTCCCTGVGLVTLLGLAVG GGGGGTGGGGCTGGTCACT CTGCTCGGCCTGGCTGTGG GC SS- SecretionATGTCGGACCTGCTACTAC 70 MSDLLLLGLIGG 132 035 signal TGGGCCTGATTGGGGGCCTLTLLLLLTLLAFA GACTCTCTTACTGCTGCTG ACGCTGCTAGCCTTTGCC SS- SecretionATGGAGACTGTGGTGATTG 71 METVVIVAIGVL 133 036 signal TTGCCATAGGTGTGCTGGCATIFLASFAALVL CACCATGTTTCTGGCTTCGT VCRQ TTGCAGCCTTGGTGCTGGT TTGCAGGCAGSS- Secretion ATGCGCGGCTCTGTGGAGT 72 MAGSVECTWGW 134 037 signalGCACCTGGGGTTGGGGGCA GHCAPSPLLLWT CTGTGCCCCCAGCCCCCTG LLLFAAPFGLLGCTCCTTTGGACTCTACTTCT GTTTGCAGCCCCATTTGGC CTGCTGGGG SS- SecretionATGATGCCGTCCCGTACCA 73 MMPSRTNLATGI 135 038 signal ACCTGGCTACTGGAATCCCPSSKVKYSRLSST CAGTAGTAAAGTGAAATAT DDGYIDLQFKKT TCAAGGCTCTCCAGCACAGPPKIPYKAIALAT ACGATGGCTACATTGACCT VLFLIGA TCAGTTTAAGAAAACCCCTCCTAAGATCCCTTATAAGG CCATCGCACTTGCCACTGT GCTGTTTTTGATTGGCGCC SS-Secretion ATGGCCCTGCCCCAGATGT 74 MALPQMCDGSH 136 039 signalGTGACGGGAGCCACTTGGC LASTLRYCMTVS CTCCACCCTCCGCTATTGC GTVVLVAGTLCFAATGACAGTCAGCGGCACAG TGGTTCTGGTGGCCGGGAC GCTCTGCTTCGCT SS- Vrg-6TGAAAAAGTGGTTCGTTGC 75 MKKWFVAAGIG 137 041 TGCCGGCATCGGCGCTGCCAGLLMLSSAA GGACTCATGCTCTCCAGCG CCGCCA SS- PhoA ATGAAACAGAGCACCATTG 76MKQSTIALALLPL 138 042 CGCTGGCGCTGCTGCCGCT LFTPVTKA GCTGTTTACCCCGGTGACCAAAGCG SS- OmpA ATGAAAAAAACCGCGATTG 77 MKKTAIAIAVAL 139 043CGATTGCGGTGGCGCTGGC AGFATVAQA GGGCTTTGCGACCGTGGCG CAGGCG SS- STIATGAAAAAACTGATGCTGG 78 MKKLMLAIFFSV 140 044 CGATTTTTTTTAGCGTGCTGLSFPSFSQS AGCTTTCCGAGCTTTAGCC AGAGC SS- STII ATGAAAAAAAACATTGCGT 79MKKNIAFLLASM 141 045 TTCTGCTGGCGAGCATGTT FVFSIATNAYA TGTGTTTAGCATTGCGACCAACGCGTATGCG SS- Amylase ATGTTTGCGAAACGCTTTA 80 MFAKRFKTSLLP 142 046AAACCAGCCTGCTGCCGCT LFAGFLLLFHLVL GTTTGCGGGCTTTCTGCTGC AGPAAASTGTTTCATCTGGTGCTGGC GGGCCCGGCGGCGGCGAGC SS- Alpha ATGCGCTTTCCGAGCATTTT81 MRFPSIFTAVLFA 143 047 Factor TACCGCGGTGCTGTTTGCG ASSALAGCGAGCAGCGCGCTGGCG SS- Alpha ATGCGCTTTCCGAGCATTTT 82 MRFPSIFTTVLFA 144048 Factor TACCACCGTGCTGTTTGCG ASSALA GCGAGCAGCGCGCTGGCG SS- AlphaATGCGCTTTCCGAGCATTTT 83 MRFPSIFTSVLFA 145 049 Factor TACCAGCGTGCTGTTTGCGASSALA GCGAGCAGCGCGCTGGCG SS- Alpha ATGCGCTTTCCGAGCATTTT 84MRFPSIFTHVLFA 146 050 Factor TACCCATGTGCTGTTTGCG ASSALAGCGAGCAGCGCGCTGGCG SS- Alpha ATGCGCTTTCCGAGCATTTT 85 MRFPSIFTIVLFA 147051 Factor TACCATTGTGCTGTTTGCG ASSALA GCGAGCAGCGCGCTGGCG SS- AlphaATGCGCTTTCCGAGCATTTT 86 MRFPSIFTFVLFA 148 052 FactorTACCTTTGTGCTGTTTGCGG ASSALA CGAGCAGCGCGCTGGCG SS- AlphaATGCGCTTTCCGAGCATTTT 87 MRFPSIFTEVLFA 149 053 Factor TACCGAAGTGCTGTTTGCGASSALA GCGAGCAGCGCGCTGGCG SS- Alpha ATGCGCTTTCCGAGCATTTT 88MRFPSIFTGVLFA 150 054 Factor TACCGGCGTGCTGTTTGCG ASSALAGCGAGCAGCGCGCTGGCG SS- Endoglucanase V ATGCGTTCCTCCCCCCTCCT 89MRSSPLLRSAVV 151 055 CCGCTCCGCCGTTGTGGCC AALPVLALA GCCCTGCCGGTGTTGGCCCTTGCC SS- Secretion ATGGGCGCGGCGGCCGTGC 90 MGAAAVRWHLC 152 056 signalGCTGGCACTTGTGCGTGCT VLLALGTRGRL GCTGGCCCTGGGCACACGC GGGCGGCTG SS- FungalATGAGGAGCTCCCTTGTGC 91 MRSSLVLFFVSA 153 057 TGTTCTTTGTCTCTGCGTGG WTALAACGGCCTTGGCCAG SS- Fibronectin ATGCTCAGGGGTCCGGGAC 92 MLRGPGPGRLLL 154058 CCGGGCGGCTGCTGCTGCT LAVLCLGTSVRC AGCAGTCCTGTGCCTGGGG TETGKSKRACATCGGTGCGCTGCACCG AAACCGGGAAGAGCAAGA GG SS- FibronectinATGCTTAGGGGTCCGGGGC 93 MLRGPGPGLLLL 155 059 CCGGGCTGCTGCTGCTGGCAVQCLGTAVPST CGTCCAGCTGGGGACAGCG GA GTGCCCTCCACG SS- FibronectinATGCGCCGGGGGGCCCTGA 94 MRRGALTGLLLV 156 060 CCGGGCTGCTCCTGGTCCTLCLSVVLRAAPS GTGCCTGAGTGTTGTGCTA ATSKKRR CGTGCAGCCCCCTCTGCAACAAGCAAGAAGCGCAGG

In the table, SS is secretion signal and MLS is mitochondrial leadersignal. The primary constructs or mmRNA of the present invention may bedesigned to encode any of the signal sequences of SEQ ID NOs 95-156, orfragments or variants thereof. These sequences may be included at thebeginning of the polypeptide coding region, in the middle or at theterminus or alternatively into a flanking region. Further, any of thepolynucleotide primary constructs of the present invention may alsocomprise one or more of the sequences defined by SEQ ID NOs 33-94. Thesemay be in the first region or either flanking region.

Additional signal sequences which may be utilized in the presentinvention include those taught in, for example, databases such as thosefound at http://www.signalpeptide.de/ orhttp://proline.bic.nus.edu.sg/spdb/. Those described in U.S. Pat. Nos.8,124,379; 7,413,875 and 7,385,034 are also within the scope of theinvention and the contents of each are incorporated herein by referencein their entirety.

Target Selection

According to the present invention, the primary constructs comprise atleast a first region of linked nucleosides encoding at least onepolypeptide of interest.

In one embodiment, the at least one polypeptide of interest may includeany polypeptide capable of signaling or acting as a binding parter toeffect expression from the accessory plasmid including but not limitedto a polypeptide of interest described in U.S. Provisional PatentApplication No. 61/618,862, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Biologics, U.S. Provisional PatentApplication No. 61/681,645, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Biologics, U.S. Provisional PatentApplication No. 61/737,130, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Biologics, International PatentApplication No. PCT/US2013/030062, filed Mar. 9, 2013, entitled ModifiedPolynucleotidse for the Production of Biologics and Proteins Associatedwith Human Disease, U.S. Provisional Patent Application No. 61/618,866,filed Apr. 2, 2012, entitled Modified Polynucleotides for the Productionof Antibodies, U.S. Provisional Patent Application No. 61/681,647, filedAug. 10, 2012, entitled Modified Polynucleotdies for the Production ofAntibodies, U.S. Provisional Patent Application No. 61/737,134, filedDec. 14, 2012, entitled Modified Polynucleotides for the Production ofAntibodies, International Patent Application No. PCT/US2013/030063,filed Mar. 9, 2013, entitled Modified Polynucleotides, U.S. ProvisionalPatent Application No. 61/618,868, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Vaccines, U.S. Provisional PatentApplication No. 61/681,648, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Vaccines, U.S. Provisional PatentApplication No. 61/737,135, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Vaccines, U.S. Provisional PatentApplication No. 61/618,870, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Therapeutic Proteins and Peptides,U.S. Provisional Patent Application No. 61/681,649, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of TherapeuticProteins and Peptides, U.S. Provisional Patent Application No.61/737,139, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Therapeutic Proteins and Peptides, U.S. ProvisionalPatent Application No. 61/618,873, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Secreted Proteins, U.S.Provisional Patent Application No. 61/681,650, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of SecretedProteins, U.S. Provisional Patent Application No. 61/737,147, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofSecreted Proteins, International Patent Application No.PCT/US2013/030064, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Secreted Proteins, U.S. Provisional PatentApplication No. 61/618,878, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Plasma Membrane Proteins, U.S.Provisional Patent Application No. 61/681,654, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Plasma MembraneProteins, U.S. Provisional Patent Application No. 61/737,152, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production of PlasmaMembrane Proteins, International Patent Application No.PCT/US2013/030059, filed Mar. 9, 2013, entitled Modified Polynucleotidesfor the Production of Membrane Proteins, U.S. Provisional PatentApplication No. 61/618,885, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins, U.S. Provisional Patent Application No. 61/681,658, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/737,155, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Cytoplasmic and CytoskeletalProteins, International Patent Application No. PCT/US2013/030066, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofCytoplasmic and Cytoskeletal Proteins, U.S. Provisional PatentApplication No. 61/618,896, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrance BoundProteins, U.S. Provisional Patent Application No. 61/668,157, filed Jul.5, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/681,661, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Intracellular Membrane BoundProteins, U.S. Provisional Patent Application No. 61/737,160, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofIntracellular Membrane Bound Proteins, U.S. Provisional PatentApplication No. 61/618,911, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Nuclear Proteins, U.S. ProvisionalPatent Application No. 61/681,667, filed Aug. 10, 2012, entitledModified Polynucleotides for the Production of Nuclear Proteins, U.S.Provisional Patent Application No. 61/737,168, filed Dec. 14, 2012,entitled Modified Polynucleotides for the Production of NuclearProteins, International Patent Application No. PCT/US2013/030067, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofNuclear Proteins, U.S. Provisional Patent Application No. 61/618,922,filed Apr. 2, 2012, entitled Modified Polynucleotides for the Productionof Proteins, U.S. Provisional Patent Application No. 61/681,675, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofProteins, U.S. Provisional Application No. 61/737,174, filed Dec. 14,2012, entitled Modified Polynucleotides for the Production of Proteins,International Patent Application No PCT/US2013/030060, filed Mar. 9,2013, entitled Modified Polynucleotides for the Production of Proteins,U.S. Provisional Patent Application No. 61/618,935, filed Apr. 2, 2012,entitled Modified Polynucleotides for the Production of ProteinAssociated with Human Disease, U.S. Provisional Patent Application No.61/681,687, filed Aug. 10, 2012, entitled Modified Polynucleotides forthe Production of Proteins Associated with Human Disease, U.S. PatentApplication No. 61/737,184, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, International Patent Application No PCT/US2013/030061, filedMar. 9, 2013, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/618,945, filed Apr. 2, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/681,696, filed Aug.10, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/737,191, filed Dec. 14, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/618,953, filed Apr.2, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, U.S. Provisional PatentApplication No. 61/681,704, filed Aug. 10, 2012, entitled ModifiedPolynucleotides for the Production of Proteins Associated with HumanDisease, U.S. Provisional Patent Application No. 61/737,203, filed Dec.14, 2012, entitled Modified Polynucleotides for the Production ofProteins Associated with Human Disease, International Patent ApplicationNo PCT/US2013/031821, filed Mar. 15, 2013, entitled In Vivo Productionof Proteins, U.S. Provisional Patent Application No. 61/681,720, filedAug. 10, 2012, entitled Modified Polynucleotides for the Production ofCosmetic Proteins and Peptides, U.S. Provisional Patent Application No.61/737,213, filed Dec. 14, 2012, entitled Modified Polynucleotides forthe Production of Cosmetic Proteins and Peptides, International PatentApplication No PCT/US2013/030068, filed Mar. 9, 2013, entitled ModifiedPolynucleotides for the Production of Cosmetic Proteins and Peptides,U.S. Provisional Patent Application No. 61/681,742, filed Aug. 10, 2012,entitled Modified Polynucleotides for the Production of Oncology-RelatedProteins and Peptides, International Patent Application NoPCT/US2013/030070, filed Mar. 9, 2012, entitled Modified Polynucleotidesfor the Production of Oncology-Related Proteins and Peptides, thecontents of each of which are herein incorporated by reference in theirentireties.

Protein Cleavage Signals and Sites

In one embodiment, the polypeptides of the present invention may includeat least one protein cleavage signal containing at least one proteincleavage site. The protein cleavage site may be located at theN-terminus, the C-terminus, at any space between the N- and theC-termini such as, but not limited to, half-way between the N- andC-termini, between the N-terminus and the half way point, between thehalf way point and the C-terminus, and combinations thereof.

The polypeptides of the present invention may include, but is notlimited to, a proprotein convertase (or prohormone convertase), thrombinor Factor Xa protein cleavage signal. Proprotein convertases are afamily of nine proteinases, comprising seven basic amino acid-specificsubtilisin-like serine proteinases related to yeast kexin, known asprohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basicamino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilasesthat cleave at non-basic residues, called subtilisin kexin isozyme 1(SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9).Non-limiting examples of protein cleavage signal amino acid sequencesare listing in Table 7. In Table 7, “X” refers to any amino acid, “n”may be 0, 2, 4 or 6 amino acids and “*” refers to the protein cleavagesite. In Table 7, SEQ ID NO: 159 refers to when n=4 and SEQ ID NO: 160refers to when n=6.

TABLE 7 Protein Cleavage Site Sequences Protein Cleavage Amino AcidSignal Cleavage Sequence SEQ ID NO Proprotein R-X-X-R* 157 convertaseR-X-K/R-R* 158 K/R-Xn-K/R* 159 or 160 Thrombin L-V-P-R*-G-S 161 L-V-P-R*162 A/F/G/I/L/T/V/M-A/F/G/ 163 I/L/T/V/W/A-P-R* Factor Xa I-E-G-R* 164I-D-G-R* 165 A-E-G-R* 166 A/F/G/I/L/T/V/M-D/E-G-R* 167

In one embodiment, the primary constructs and the mmRNA of the presentinvention may be engineered such that the primary construct or mmRNAcontains at least one encoded protein cleavage signal. The encodedprotein cleavage signal may be located before the start codon, after thestart codon, before the coding region, within the coding region such as,but not limited to, half way in the coding region, between the startcodon and the half way point, between the half way point and the stopcodon, after the coding region, before the stop codon, between two stopcodons, after the stop codon and combinations thereof.

In one embodiment, the primary constructs or mmRNA of the presentinvention may include at least one encoded protein cleavage signalcontaining at least one protein cleavage site. The encoded proteincleavage signal may include, but is not limited to, a proproteinconvertase (or prohormone convertase), thrombin and/or Factor Xa proteincleavage signal. One of skill in the art may use Table 1 above or otherknown methods to determine the appropriate encoded protein cleavagesignal to include in the primary constructs or mmRNA of the presentinvention. For example, starting with the signal of Table 7 andconsidering the codons of Table 1 one can design a signal for theprimary construct which can produce a protein signal in the resultingpolypeptide.

In one embodiment, the polypeptides of the present invention include atleast one protein cleavage signal and/or site.

As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Pub. No.20090227660, herein incorporated by reference in their entireties, use afurin cleavage site to cleave the N-terminal methionine of GLP-1 in theexpression product from the Golgi apparatus of the cells. In oneembodiment, the polypeptides of the present invention include at leastone protein cleavage signal and/or site with the proviso that thepolypeptide is not GLP-1.

In one embodiment, the primary constructs or mmRNA of the presentinvention includes at least one encoded protein cleavage signal and/orsite.

In one embodiment, the primary constructs or mmRNA of the presentinvention includes at least one encoded protein cleavage signal and/orsite with the proviso that the primary construct or mmRNA does notencode GLP-1.

In one embodiment, the primary constructs or mmRNA of the presentinvention may include more than one coding region. Where multiple codingregions are present in the primary construct or mmRNA of the presentinvention, the multiple coding regions may be separated by encodedprotein cleavage sites. As a non-limiting example, the primary constructor mmRNA may be signed in an ordered pattern. On such pattern followsAXBY form where A and B are coding regions which may be the same ordifferent coding regions and/or may encode the same or differentpolypeptides, and X and Y are encoded protein cleavage signals which mayencode the same or different protein cleavage signals. A second suchpattern follows the form AXYBZ where A and B are coding regions whichmay be the same or different coding regions and/or may encode the sameor different polypeptides, and X, Y and Z are encoded protein cleavagesignals which may encode the same or different protein cleavage signals.A third pattern follows the form ABXCY where A, B and C are codingregions which may be the same or different coding regions and/or mayencode the same or different polypeptides, and X and Y are encodedprotein cleavage signals which may encode the same or different proteincleavage signals.

In one embodiment, the polypeptides, primary constructs and mmRNA canalso contain sequences that encode protein cleavage sites so that thepolypeptides, primary constructs and mmRNA can be released from acarrier region or a fusion partner by treatment with a specific proteasefor said protein cleavage site.

III. MODIFICATIONS

Herein, in a polynucleotide (such as a primary construct or an mRNAmolecule), the terms “modification” or, as appropriate, “modified” referto modification with respect to A, G, U or C ribonucleotides and/orNTPs. Generally, herein, these terms are not intended to refer to theribonucleotide modifications in naturally occurring 5′-terminal mRNA capmoieties. In a polypeptide, the term “modification” refers to amodification as compared to the canonical set of 20 amino acids, moiety)

The modifications may be various distinct modifications. In someembodiments, the coding region, the flanking regions and/or the terminalregions may contain one, two, or more (optionally different) nucleosideor nucleotide modifications. In some embodiments, a modifiedpolynucleotide, primary construct, or mmRNA introduced to a cell mayexhibit reduced degradation in the cell, as compared to an unmodifiedpolynucleotide, primary construct, or mmRNA.

The polynucleotides, primary constructs, and mmRNA can include anyuseful modification, such as to the sugar, the nucleobase, or theinternucleoside linkage (e.g. to a linking phosphate/to a phosphodiesterlinkage/to the phosphodiester backbone). One or more atoms of apyrimidine nucleobase may be replaced or substituted with optionallysubstituted amino, optionally substituted thiol, optionally substitutedalkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). Incertain embodiments, modifications (e.g., one or more modifications) arepresent in each of the sugar and the internucleoside linkage.Modifications according to the present invention may be modifications ofribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threosenucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids(PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additionalmodifications are described herein.

As described herein, the polynucleotides, primary constructs, and mmRNAof the invention do not substantially induce an innate immune responseof a cell into which the mRNA is introduced. Featues of an inducedinnate immune response include 1) increased expression ofpro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I,MDA5, etc, and/or 3) termination or reduction in protein translation.

In certain embodiments, it may desirable to intracellularly degrade amodified nucleic acid molecule introduced into the cell. For example,degradation of a modified nucleic acid molecule may be preferable ifprecise timing of protein production is desired. Thus, in someembodiments, the invention provides a modified nucleic acid moleculecontaining a degradation domain, which is capable of being acted on in adirected manner within a cell. In another aspect, the present disclosureprovides polynucleotides comprising a nucleoside or nucleotide that candisrupt the binding of a major groove interacting, e.g. binding, partnerwith the polynucleotide (e.g., where the modified nucleotide hasdecreased binding affinity to major groove interacting partner, ascompared to an unmodified nucleotide).

The polynucleotides, primary constructs, and mmRNA can optionallyinclude other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs,shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAsthat induce triple helix formation, aptamers, vectors, etc.). In someembodiments, the polynucleotides, primary constructs, or mmRNA mayinclude one or more messenger RNAs (mRNAs) and one or more modifiednucleoside or nucleotides (e.g., mmRNA molecules). Details for thesepolynucleotides, primary constructs, and mmRNA follow.

Polynucleotides and Primary Constructs

The polynucleotides, primary constructs, and mmRNA of the inventionincludes a first region of linked nucleosides encoding a polypeptide ofinterest, a first flanking region located at the 5′ terminus of thefirst region, and a second flanking region located at the 3′ terminus ofthe first region.

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having Formula (Ia) orFormula (Ia-1):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

- - - is a single bond or absent;

each of R^(1′), R^(2′), R^(1″), R^(2″), R¹, R², R³, R⁴, and R⁵ is,independently, if present, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, orabsent; wherein the combination of R³ with one or more of R^(1′),R^(1″), R^(2′), R^(2″), or R⁵ (e.g., the combination of R^(1′) and R³,the combination of R^(1″) and R³, the combination of R^(2′) and R³, thecombination of R^(2″) and R³, or the combination of R⁵ and R³) can jointogether to form optionally substituted alkylene or optionallysubstituted heteroalkylene and, taken together with the carbons to whichthey are attached, provide an optionally substituted heterocyclyl (e.g.,a bicyclic, tricyclic, or tetracyclic heterocyclyl); wherein thecombination of R⁵ with one or more of R^(1′), R^(1″), R^(2′), or R^(2″)(e.g., the combination of R^(1′) and R⁵, the combination of R^(1″) andR⁵, the combination of R^(2′) and R⁵, or the combination of R^(2″) andR⁵) can join together to form optionally substituted alkylene oroptionally substituted heteroalkylene and, taken together with thecarbons to which they are attached, provide an optionally substitutedheterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl);and wherein the combination of R⁴ and one or more of R^(1′), R^(1″),R^(2′), R^(2″), R³, or R⁵ can join together to form optionallysubstituted alkylene or optionally substituted heteroalkylene and, takentogether with the carbons to which they are attached, provide anoptionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl); each of m′ and m″ is, independently, aninteger from 0 to 3 (e.g., from 0 to 2, from 0 to 1, from 1 to 3, orfrom 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof), wherein the combination of B and R^(1′), the combination of Band R^(2′), the combination of B and R^(1″), or the combination of B andR^(2″) can, taken together with the carbons to which they are attached,optionally form a bicyclic group (e.g., a bicyclic heterocyclyl) orwherein the combination of B, R^(1″), and R³ or the combination of B,R^(2″), and R³ can optionally form a tricyclic or tetracyclic group(e.g., a tricyclic or tetracyclic heterocyclyl, such as in Formula(IIo)-(IIp) herein), In some embodiments, the polynucleotide, primaryconstruct, or mmRNA includes a modified ribose. In some embodiments, thepolynucleotide, primary construct, or mmRNA (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides having Formula (Ia-2)-(Ia-5) or a pharmaceuticallyacceptable salt or stereoisomer thereof

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, the first flanking region, or the secondflanking region) includes n number of linked nucleosides having Formula(Ib) or Formula (Ib-1):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

- - - is a single bond or absent;

each of R¹, R^(3′), R^(3″), and R⁴ is, independently, H, halo, hydroxy,optionally substituted alkyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionallysubstituted hydroxyalkoxy, optionally substituted amino, azido,optionally substituted aryl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, or absent; and wherein the combination of R¹ and R^(3′) orthe combination of R¹ and R^(3″) can be taken together to formoptionally substituted alkylene or optionally substituted heteroalkylene(e.g., to produce a locked nucleic acid);

each R⁵ is, independently, H, halo, hydroxy, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, or absent;

each of Y¹, Y², and Y³ is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted alkoxyalkoxy, or optionally substituted amino;

n is an integer from 1 to 100,000; and

B is a nucleobase.

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having Formula (Ic):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

- - - is a single bond or absent;

each of B¹, B², and B³ is, independently, a nucleobase (e.g., a purine,a pyrimidine, or derivatives thereof, as described herein), H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl, wherein one and only one of B¹, B²,and B³ is a nucleobase;

each of R^(b1), R^(b2), R^(b3), R³, and R⁵ is, independently, H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl oroptionally substituted aminoalkynyl;

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

wherein the ring including U can include one or more double bonds.

In particular embodiments, the ring including U does not have a doublebond between U—CB³R^(b3) or between CB³R^(b3)—C^(B2)R^(b2).

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having Formula (Id):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

U is O, S, N(R^(U))_(nu), or C(R^(U))_(nu), wherein nu is an integerfrom 0 to 2 and each R^(U) is, independently, H, halo, or optionallysubstituted alkyl;

each R³ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl;

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl, oroptionally substituted aryl;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, optionally substituted alkylene (e.g.,methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having Formula (Ie):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of U′ and U″ is, independently, O, S, N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(U) is,independently, H, halo, or optionally substituted alkyl;

each R⁶ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkynyl;

each Y^(5′) is, independently, O, S, optionally substituted alkylene(e.g., methylene or ethylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, first flanking region, or second flankingregion) includes n number of linked nucleosides having Formula (If) or(If-1):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of U′ and U″ is, independently, O, S, N, N(R^(U))_(nu), orC(R^(U))_(nu), wherein nu is an integer from 0 to 2 and each R^(U) is,independently, H, halo, or optionally substituted alkyl (e.g., U′ is Oand U″ is N);

- - - is a single bond or absent;

each of R^(1′), R^(2′), R^(1″), R^(2″), R³, and R⁴ is, independently, H,halo, hydroxy, thiol, optionally substituted alkyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted aminoalkoxy, optionallysubstituted alkoxyalkoxy, optionally substituted hydroxyalkoxy,optionally substituted amino, azido, optionally substituted aryl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, or absent; and wherein thecombination of R^(1′) and R³, the combination of R^(1″) and R³, thecombination of R^(2′) and R³, or the combination of R^(2″) and R³ can betaken together to form optionally substituted alkylene or optionallysubstituted heteroalkylene (e.g., to produce a locked nucleic acid);each of m′ and m″ is, independently, an integer from 0 to 3 (e.g., from0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);

each of Y¹, Y², and Y³, is, independently, O, S, Se, —NR^(N1)—,optionally substituted alkylene, or optionally substitutedheteroalkylene, wherein R^(N1) is H, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, or absent;

each Y⁴ is, independently, H, hydroxy, thiol, boranyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, optionally substituted alkynyloxy, optionallysubstituted thioalkoxy, optionally substituted alkoxyalkoxy, oroptionally substituted amino;

each Y⁵ is, independently, O, S, Se, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene;

n is an integer from 1 to 100,000; and

B is a nucleobase (e.g., a purine, a pyrimidine, or derivativesthereof).

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If), and (IIa)-(IIp)), thering including U has one or two double bonds.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachof R¹, R^(1′), and R^(1″), if present, is H. In further embodiments,each of R², R^(2′), and R^(2″), if present, is, independently, H, halo(e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In particularembodiments, alkoxyalkoxy is —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl). In some embodiments, s2 is 0, s1 is 1 or 2,s3 is 0 or 1, and R′ is C₁₋₆ alkyl.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachof R², R^(2′), and R^(2″), if present, is H. In further embodiments,each of R¹, R^(1′), and R^(1″), if present, is, independently, H, halo(e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In particularembodiments, alkoxyalkoxy is —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl). In some embodiments, s2 is 0, s1 is 1 or 2,s3 is 0 or 1, and R′ is C₁₋₆ alkyl.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachof R³, R⁴, and R⁵ is, independently, H, halo (e.g., fluoro), hydroxy,optionally substituted alkyl, optionally substituted alkoxy (e.g.,methoxy or ethoxy), or optionally substituted alkoxyalkoxy. Inparticular embodiments, R³ is H, R⁴ is H, R⁵ is H, or R³, R⁴, and R⁵ areall H. In particular embodiments, R³ is C₁₋₆ alkyl, R⁴ is C₁₋₆ alkyl, R⁵is C₁₋₆ alkyl, or R³, R⁴, and R⁵ are all C₁₋₆ alkyl. In particularembodiments, R³ and R⁴ are both H, and R⁵ is C₁₋₆ alkyl.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R³ andR⁵ join together to form optionally substituted alkylene or optionallysubstituted heteroalkylene and, taken together with the carbons to whichthey are attached, provide an optionally substituted heterocyclyl (e.g.,a bicyclic, tricyclic, or tetracyclic heterocyclyl, such as trans-3′,4′analogs, wherein R³ and R⁵ join together to form heteroalkylene (e.g.,—(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein each of b1, b2, and b3 are,independently, an integer from 0 to 3).

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R³ andone or more of R^(1′), R^(1″), R^(2′), R^(2″), or R⁵ join together toform optionally substituted alkylene or optionally substitutedheteroalkylene and, taken together with the carbons to which they areattached, provide an optionally substituted heterocyclyl (e.g., abicyclic, tricyclic, or tetracyclic heterocyclyl, R³ and one or more ofR^(1′), R^(1″), R^(2″), or R⁵ join together to form heteroalkylene(e.g., —(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein each of b1, b2, andb3 are, independently, an integer from 0 to 3).

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R⁵ andone or more of R^(1′), R^(1″), R^(2′), or R^(2″) join together to formoptionally substituted alkylene or optionally substituted heteroalkyleneand, taken together with the carbons to which they are attached, providean optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, ortetracyclic heterocyclyl, R⁵ and one or more of R^(1′), R^(1″), R^(2′),or R^(2″) join together to form heteroalkylene (e.g.,—(CH₂)_(b1)O(CH₂)_(b2)O(CH₂)_(b3)—, wherein each of b1, b2, and b3 are,independently, an integer from 0 to 3).

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachY² is, independently, O, S, or —NR^(N1)—, wherein R^(N1) is H,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl. In particularembodiments, Y² is NR^(N1)—, wherein R^(N1) is H or optionallysubstituted alkyl (e.g., C1-6 alkyl, such as methyl, ethyl, isopropyl,or n-propyl).

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachY³ is, independently, O or S.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R¹ isH; each R² is, independently, H, halo (e.g., fluoro), hydroxy,optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionallysubstituted alkoxyalkoxy (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is0 or 1, and R′ is C₁₋₆ alkyl); each Y² is, independently, O or—NR^(N1)—, wherein R^(N1) is H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, or optionallysubstituted aryl (e.g., wherein R^(N1) is H or optionally substitutedalkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl, orn-propyl)); and each Y³ is, independently, 0 or S (e.g., S). In furtherembodiments, R³ is H, halo (e.g., fluoro), hydroxy, optionallysubstituted alkyl, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In yet furtherembodiments, each Y¹ is, independently, O or —NR^(N1)—, wherein R^(N1)is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, or optionally substituted aryl (e.g.,wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆ alkyl,such as methyl, ethyl, isopropyl, or n-propyl)); and each Y⁴ is,independently, H, hydroxy, thiol, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted thioalkoxy,optionally substituted alkoxyalkoxy, or optionally substituted amino.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), eachR¹ is, independently, H, halo (e.g., fluoro), hydroxy, optionallysubstituted alkoxy (e.g., methoxy or ethoxy), or optionally substitutedalkoxyalkoxy (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each ofs2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H orC₁₋₂₀ alkyl, such as wherein s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R′is C₁₋₆ alkyl); R² is H; each Y² is, independently, O or —NR^(N1)—,wherein R^(N1) is H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, or optionallysubstituted aryl (e.g., wherein R^(N1) is H or optionally substitutedalkyl (e.g., C₁₋₆ alkyl, such as methyl, ethyl, isopropyl, orn-propyl)); and each Y³ is, independently, O or S (e.g., S). In furtherembodiments, R³ is H, halo (e.g., fluoro), hydroxy, optionallysubstituted alkyl, optionally substituted alkoxy (e.g., methoxy orethoxy), or optionally substituted alkoxyalkoxy. In yet furtherembodiments, each Y¹ is, independently, O or —NR^(N1)—, wherein R^(N1)is H, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, or optionally substituted aryl (e.g.,wherein R^(N1) is H or optionally substituted alkyl (e.g., C₁₋₆ alkyl,such as methyl, ethyl, isopropyl, or n-propyl)); and each Y⁴ is,independently, H, hydroxy, thiol, optionally substituted alkyl,optionally substituted alkoxy, optionally substituted thioalkoxy,optionally substituted alkoxyalkoxy, or optionally substituted amino.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), thering including U is in the β-D (e.g., β-D-ribo) configuration.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), thering including U is in the α-L (e.g., α-L-ribo) configuration.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), one ormore B is not pseudouridine (ψ) or 5-methyl-cytidine (m⁵C). In someembodiments, about 10% to about 100% of n number of B nucleobases is notψ or m⁵C (e.g., from 10% to 20%, from 10% to 35%, from 10% to 50%, from10% to 60%, from 10% to 75%, from 10% to 90%, from 10% to 95%, from 10%to 98%, from 10% to 99%, from 20% to 35%, from 20% to 50%, from 20% to60%, from 20% to 75%, from 20% to 90%, from 20% to 95%, from 20% to 98%,from 20% to 99%, from 20% to 100%, from 50% to 60%, from 50% to 75%,from 50% to 90%, from 50% to 95%, from 50% to 98%, from 50% to 99%, from50% to 100%, from 75% to 90%, from 75% to 95%, from 75% to 98%, from 75%to 99%, and from 75% to 100% of n number of B is not ψ or m⁵C). In someembodiments, B is not ψ or m⁵C.

In some embodiments of the polynucleotides, primary constructs, or mmRNA(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2),(IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), when Bis an unmodified nucleobase selected from cytosine, guanine, uracil andadenine, then at least one of Y¹, Y², or Y³ is not O.

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a modified ribose. In some embodiments, the polynucleotide,primary construct, or mmRNA (e.g., the first region, the first flankingregion, or the second flanking region) includes n number of linkednucleosides having Formula (IIa)-(IIc):

or a pharmaceutically acceptable salt or stereoisomer thereof. Inparticular embodiments, U is O or C(R^(U))_(nu), wherein nu is aninteger from 0 to 2 and each R^(U) is, independently, H, halo, oroptionally substituted alkyl (e.g., U is —CH₂— or —CH—). In otherembodiments, each of R¹, R², R³, R⁴, and R⁵ is, independently, H, halo,hydroxy, thiol, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted alkenyloxy, optionally substitutedalkynyloxy, optionally substituted aminoalkoxy, optionally substitutedalkoxyalkoxy, optionally substituted hydroxyalkoxy, optionallysubstituted amino, azido, optionally substituted aryl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, or absent (e.g., each R¹ and R² is,independently, H, halo, hydroxy, optionally substituted alkyl, oroptionally substituted alkoxy; each R³ and R⁴ is, independently, H oroptionally substituted alkyl; and R⁵ is H or hydroxy), and

is a single bond or double bond. In particular embodiments, thepolynucleotidesor mmRNA includes n number of linked nucleosides havingFormula (IIb-1)-(IIb-2):

or a pharmaceutically acceptable salt or stereoisomer thereof. In someembodiments, U is 0 or C(R^(U))_(nu), wherein nu is an integer from 0 to2 and each R^(U) is, independently, H, halo, or optionally substitutedalkyl (e.g., U is —CH₂— or —CH—). In other embodiments, each of R¹ andR² is, independently, H, halo, hydroxy, thiol, optionally substitutedalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkoxy,optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, or absent(e.g., each R¹ and R² is, independently, H, halo, hydroxy, optionallysubstituted alkyl, or optionally substituted alkoxy, e.g., H, halo,hydroxy, alkyl, or alkoxy). In particular embodiments, R² is hydroxy oroptionally substituted alkoxy (e.g., methoxy, ethoxy, or any describedherein).

In particular embodiments, the polynucleotide, primary construct, ormmRNA includes n number of linked nucleosides having Formula(IIc-1)-(IIc-4):

or a pharmaceutically acceptable salt or stereoisomer thereof. In someembodiments, U is O or C(R^(U))_(nu), wherein nu is an integer from 0 to2 and each R^(U) is, independently, H, halo, or optionally substitutedalkyl (e.g., U is —CH₂— or —CH—). In some embodiments, each of R¹, R²,and R³ is, independently, H, halo, hydroxy, thiol, optionallysubstituted alkyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedhydroxyalkoxy, optionally substituted amino, azido, optionallysubstituted aryl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, optionally substituted aminoalkynyl, or absent(e.g., each R¹ and R² is, independently, H, halo, hydroxy, optionallysubstituted alkyl, or optionally substituted alkoxy, e.g., H, halo,hydroxy, alkyl, or alkoxy; and each R³ is, independently, H oroptionally substituted alkyl)). In particular embodiments, R² isoptionally substituted alkoxy (e.g., methoxy or ethoxy, or any describedherein). In particular embodiments, R¹ is optionally substituted alkyl,and R² is hydroxy. In other embodiments, R¹ is hydroxy, and R² isoptionally substituted alkyl. In further embodiments, R³ is optionallysubstituted alkyl.

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes an acyclic modified ribose. In some embodiments, thepolynucleotide, primary construct, or mmRNA (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides having Formula (IId)-(IIf):

or a pharmaceutically acceptable salt or stereoisomer thereof

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes an acyclic modified hexitol. In some embodiments, thepolynucleotide, primary construct, or mmRNA (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides Formula (IIg)-(IIj):

or a pharmaceutically acceptable salt or stereoisomer thereof

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a sugar moiety having a contracted or an expanded ribose ring.In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, the first flanking region, or the secondflanking region) includes n number of linked nucleosides having Formula

(IIk)-(IIm):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach of R^(1′), R″, R^(2′), and R^(2″) is, independently, H, halo,hydroxy, optionally substituted alkyl, optionally substituted alkoxy,optionally substituted alkenyloxy, optionally substituted alkynyloxy,optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy,or absent; and wherein the combination of R^(2′) and R³ or thecombination of R^(2″) and R³ can be taken together to form optionallysubstituted alkylene or optionally substituted heteroalkylene.

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a locked modified ribose. In some embodiments, thepolynucleotide, primary construct, or mmRNA (e.g., the first region, thefirst flanking region, or the second flanking region) includes n numberof linked nucleosides having Formula (IIn):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(3′) is O, S, or —NR^(N1)—, wherein R^(N1) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl and R^(3″) isoptionally substituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)or optionally substituted heteroalkylene (e.g., —CH₂NH—, —CH₂CH₂NH—,—CH₂OCH₂—, or —CH₂CH₂OCH₂—)(e.g., R^(3′) is O and R^(3″) is optionallysubstituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)).

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes n number of linked nucleosides having Formula (IIn-1)-(IIn-2):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(3′) is O, S, or —NR^(N1)—, wherein R^(N1) is H, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted aryl and R^(3″) isoptionally substituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)or optionally substituted heteroalkylene (e.g., —CH₂NH—, —CH₂CH₂NH—,—CH₂OCH₂—, or —CH₂CH₂OCH₂—) (e.g., R^(3′) is O and R^(3″) is optionallysubstituted alkylene (e.g., —CH₂—, —CH₂CH₂—, or —CH₂CH₂CH₂—)).

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a locked modified ribose that forms a tetracyclic heterocyclyl.In some embodiments, the polynucleotide, primary construct, or mmRNA(e.g., the first region, the first flanking region, or the secondflanking region) includes n number of linked nucleosides having Formula(IIo)-(IIp):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR^(12a), R^(12c), T^(1′), T^(1″), T^(2′), T^(2″), V¹, and V³ are asdescribed herein.

Any of the formulas for the polynucleotides, primary constructs, ormmRNA can include one or more nucleobases described herein (e.g.,Formulas (b1)-(b43)).

In one embodiment, the present invention provides methods of preparing apolynucleotide, primary construct, or mmRNA, wherein the polynucleotidecomprises n number of nucleosides having Formula (Ia), as definedherein:

the method comprising reacting a compound of Formula (IIIa), as definedherein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a polynucleotide, primary construct, or mmRNA, the methodscomprising: reacting a compound of Formula (IIIa), as defined herein,with a primer, a cDNA template, and an RNA polymerase.

In one embodiment, the present invention provides methods of preparing apolynucleotide, primary construct, or mmRNA, wherein the polynucleotidecomprises n number of nucleosides having Formula (Ia-1), as definedherein:

the method comprising reacting a compound of Formula (IIIa-1), asdefined herein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a polynucleotide, primary construct, or mmRNA, the methodcomprising: reacting a compound of Formula (IIIa-1), as defined herein,with a primer, a cDNA template, and an RNA polymerase.

In one embodiment, the present invention provides methods of preparing amodified mRNA, wherein the polynucleotide comprises n number ofnucleosides having Formula (Ia-2), as defined herein:

the method comprising reacting a compound of Formula (IIIa-2), asdefined herein:

with an RNA polymerase, and a cDNA template.

In a further embodiment, the present invention provides methods ofamplifying a modified mRNA, the method comprising: reacting a compoundof Formula (IIIa-2), as defined herein, with a primer, a cDNA template,and an RNA polymerase.

In some embodiments, the reaction may be repeated from 1 to about 7,000times. In any of the embodiments herein, B may be a nucleobase ofFormula (b1)-(b43).

The polynucleotides, primary constructs, and mmRNA can optionallyinclude 5′ and/or 3′ flanking regions, which are described herein.

Modified RNA (mmRNA) Molecules

The present invention also includes building blocks, e.g., modifiedribonucleosides, modified ribonucleotides, of modified RNA (mmRNA)molecules. For example, these building blocks can be useful forpreparing the polynucleotides, primary constructs, or mmRNA of theinvention.

In some embodiments, the building block molecule has Formula (IIIa) or(IIIa-1):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinthe substituents are as described herein (e.g., for Formula (Ia) and(Ia-1)), and wherein when B is an unmodified nucleobase selected fromcytosine, guanine, uracil and adenine, then at least one of Y¹, Y², orY³ is not O.

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IVa)-(IVb):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, Formula (IVa) or (IVb) is combined with a modified uracil(e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), suchas formula (b1), (b8), (b28), (b29), or (b30)). In particularembodiments, Formula (IVa) or (IVb) is combined with a modified cytosine(e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36),such as formula (b10) or (b32)). In particular embodiments, Formula(IVa) or (IVb) is combined with a modified guanine (e.g., any one offormulas (b15)-(b17) and (b37)-(b40)). In particular embodiments,Formula (IVa) or (IVb) is combined with a modified adenine (e.g., anyone of formulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IVc)-(IV1):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IVc)-(IV1) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IVc)-(IV1) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IVc)-(IV1) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IVc)-(IV1) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (Va) or (Vb):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IXa)-(IXd):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXa)-(IXd) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXa)-(IXd) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXa)-(IXd) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXa)-(IXd) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IXe)-(IXg):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXe)-(IXg) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXe)-(IXg) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXe)-(IXg) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXe)-(IXg) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IXh)-(IXk):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Bis as described herein (e.g., any one of (b1)-(b43)). In particularembodiments, one of Formulas (IXh)-(IXk) is combined with a modifieduracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and(b28)-(b31), such as formula (b1), (b8), (b28), (b29), or (b30)). Inparticular embodiments, one of Formulas (IXh)-(IXk) is combined with amodified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25),and (b32)-(b36), such as formula (b10) or (b32)). In particularembodiments, one of Formulas (IXh)-(IXk) is combined with a modifiedguanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). Inparticular embodiments, one of Formulas (IXh)-(IXk) is combined with amodified adenine (e.g., any one of formulas (b18)-(b20) and(b41)-(b43)).

In other embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, hasFormula (IXl)-(IXr):

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r1 and r2 is, independently, an integer from 0 to 5 (e.g., from 0to 3, from 1 to 3, or from 1 to 5) and B is as described herein (e.g.,any one of (b1)-(b43)). In particular embodiments, one of Formulas(IXl)-(IXr) is combined with a modified uracil (e.g., any one offormulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (b1),(b8), (b28), (b29), or (b30)). In particular embodiments, one ofFormulas (IXl)-(IXr) is combined with a modified cytosine (e.g., any oneof formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula(b10) or (b32)). In particular embodiments, one of Formulas (IXl)-(IXr)is combined with a modified guanine (e.g., any one of formulas(b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas(IXl)-(IXr) is combined with a modified adenine (e.g., any one offormulas (b18)-(b20) and (b41)-(b43)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can beselected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can beselected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5) and s1 is as described herein.

In some embodiments, the building block molecule, which may beincorporated into a nucleic acid (e.g., RNA, mRNA, polynucleotide,primary construct, or mmRNA), is a modified uridine (e.g., selected fromthe group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, is amodified cytidine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)). For example, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, is amodified adenosine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY¹, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, is amodified guanosine (e.g., selected from the group consisting of:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereinY′, Y³, Y⁴, Y⁶, and r are as described herein (e.g., each r is,independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3,or from 1 to 5)).

In some embodiments, the chemical modification can include replacementof C group at C-5 of the ring (e.g., for a pyrimidine nucleoside, suchas cytosine or uracil) with N (e.g., replacement of the >CH group at C-5with >NR^(N1) group, wherein R^(N)1 is H or optionally substitutedalkyl). For example, the building block molecule, which may beincorporated into a polynucleotide, primary construct, or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In another embodiment, the chemical modification can include replacementof the hydrogen at C-5 of cytosine with halo (e.g., Br, Cl, F, or I) oroptionally substituted alkyl (e.g., methyl). For example, the buildingblock molecule, which may be incorporated into a polynucleotide, primaryconstruct, or mmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

In yet a further embodiment, the chemical modification can include afused ring that is formed by the NH2 at the C-4 position and the carbonatom at the C-5 position. For example, the building block molecule,which may be incorporated into a polynucleotide, primary construct, ormmRNA, can be:

or a pharmaceutically acceptable salt or stereoisomer thereof, whereineach r is, independently, an integer from 0 to 5 (e.g., from 0 to 3,from 1 to 3, or from 1 to 5).

Modifications on the Sugar

The modified nucleosides and nucleotides (e.g., building blockmolecules), which may be incorporated into a polynucleotide, primaryconstruct, or mmRNA (e.g., RNA or mRNA, as described herein), can bemodified on the sugar of the ribonucleic acid. For example, the 2′hydroxyl group (OH) can be modified or replaced with a number ofdifferent substituents. Exemplary substitutions at the 2′-positioninclude, but are not limited to, H, halo, optionally substituted C₁₋₆alkyl; optionally substituted C₁₋₆ alkoxy; optionally substituted C₆₋₁₀aryloxy; optionally substituted C₃₋₈ cycloalkyl; optionally substitutedC₃₋₈ cycloalkoxy; optionally substituted C₆₋₁₀ aryloxy; optionallysubstituted C₆₋₁₀ aryl-C₁₋₆ alkoxy, optionally substituted C₁₋₁₂(heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any describedherein); a polyethyleneglycol (PEG), —O(CH₂CH₂O)_(n)CH₂CH₂OR, where R isH or optionally substituted alkyl, and n is an integer from 0 to 20(e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4,from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA)in which the 2′-hydroxyl is connected by a C₁₋₆ alkylene or C₁₋₆heteroalkylene bridge to the 4′-carbon of the same ribose sugar, whereexemplary bridges included methylene, propylene, ether, or aminobridges; aminoalkyl, as defined herein; aminoalkoxy, as defined herein;amino as defined herein; and amino acid, as defined herein

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting modified nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as foranhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, andmorpholino that also has a phosphoramidate backbone); multicyclic forms(e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA)(e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attachedto phosphodiester bonds), threose nucleic acid (TNA, where ribose isreplace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA,where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a polynucleotide, primaryconstruct, or mmRNA molecule can include nucleotides containing, e.g.,arabinose, as the sugar.

Modifications on the Nucleobase

The present disclosure provides for modified nucleosides andnucleotides. As described herein “nucleoside” is defined as a compoundcontaining a sugar molecule (e.g., a pentose or ribose) or a derivativethereof in combination with an organic base (e.g., a purine orpyrimidine) or a derivative thereof (also referred to herein as“nucleobase”). As described herein, “nucleotide” is defined as anucleoside including a phosphate group. In some embodiments, thenucleosides and nucleotides described herein are generally chemicallymodified. Exemplary non-limiting modifications include an amino group, athiol group, an alkyl group, a halo group, or any described herein. Themodified nucleotides may by synthesized by any useful method, asdescribed herein (e.g., chemically, enzymatically, or recombinantly toinclude one or more modified or non-natural nucleosides).

The modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil.

The modified nucleosides and nucleotides can include a modifiednucleobase. Examples of nucleobases found in RNA include, but are notlimited to, adenine, guanine, cytosine, and uracil. Examples ofnucleobase found in DNA include, but are not limited to, adenine,guanine, cytosine, and thymine. These nucleobases can be modified orwholly replaced to provide polynucleotides, primary constructs, or mmRNAmolecules having enhanced properties, e.g., resistance to nucleasesthrough disruption of the binding of a major groove binding partner.Table 8 below identifies the chemical faces of each canonicalnucleotide. Circles identify the atoms comprising the respectivechemical regions.

TABLE 8 Major Groove Face Minor Groove Face Watson-Crick Base-pairingFace Pyrim- idines Cytidine:

Uridine:

Purines Adenosine:

Guanosine:

In some embodiments, B is a modified uracil. Exemplary modified uracilsinclude those having Formula (b1)-(b5):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

is a single or double bond;

each of T^(1′), T^(1″), T^(2′), and T^(2″) is, independently, H,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy, or the combination of T^(1′) andT^(1″) or the combination of T^(2′) and T^(2″) join together (e.g., asin T²) to form O (oxo), S (thio), or Se (seleno);

each of V¹ and V² is, independently, O, S, N(R^(Vb))_(nv), orC(R^(Vb))_(nv), wherein nv is an integer from 0 to 2 and each R^(Vb) is,independently, H, halo, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,optionally substituted aminoalkyl (e.g., substituted with anN-protecting group, such as any described herein, e.g.,trifluoroacetyl), optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g.,substituted with an N-protecting group, such as any described herein,e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, or optionally substituted alkynyloxy (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl);

R¹⁰ is H, halo, optionally substituted amino acid, hydroxy, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aminoalkyl, optionallysubstituted hydroxyalkyl, optionally substituted hydroxyalkenyl,optionally substituted hydroxyalkynyl, optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted alkoxy, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,optionally substituted carboxyalkoxy, optionally substitutedcarboxyalkyl, or optionally substituted carbamoylalkyl;

R¹¹ is H or optionally substituted alkyl;

R^(12a) is H, optionally substituted alkyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, or optionally substitutedaminoalkynyl, optionally substituted carboxyalkyl (e.g., optionallysubstituted with hydroxy), optionally substituted carboxyalkoxy,optionally substituted carboxyaminoalkyl, or optionally substitutedcarbamoylalkyl; and

R^(12c) is H, halo, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, optionally substituted amino,optionally substituted hydroxyalkyl, optionally substitutedhydroxyalkenyl, optionally substituted hydroxyalkynyl, optionallysubstituted aminoalkyl, optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl.

Other exemplary modified uracils include those having Formula (b6)-(b9):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

is a single or double bond;

each of T^(1′), T^(1″), T^(2′), and T^(2″) is, independently, H,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy, or the combination of T^(1′) andT^(1″) join together (e.g., as in T¹) or the combination of T^(2′) andT^(2″) join together (e.g., as in T²) to form O (oxo), S (thio), or Se(seleno), or each T¹ and T² is, independently, O (oxo), S (thio), or Se(seleno);

each of W¹ and W² is, independently, N(R^(Wa))_(nw) or C(R^(Va)a)_(nw),wherein nw is an integer from 0 to 2 and each R^(Wa) is, independently,H, optionally substituted alkyl, or optionally substituted alkoxy;

each V³ is, independently, O, S, N(R^(Va))_(nv), or C(R^(Va))_(nv),wherein nv is an integer from 0 to 2 and each R^(Va) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted hydroxyalkyl, optionally substitutedhydroxyalkenyl, optionally substituted hydroxyalkynyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted heterocyclyl, optionally substituted alkheterocyclyl,optionally substituted alkoxy, optionally substituted alkenyloxy, oroptionally substituted alkynyloxy, optionally substituted aminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted acylaminoalkyl (e.g., substituted with an N-protectinggroup, such as any described herein, e.g., trifluoroacetyl), optionallysubstituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl), and wherein R^(V)a and R¹²Ctaken together with the carbon atoms to which they are attached can formoptionally substituted cycloalkyl, optionally substituted aryl, oroptionally substituted heterocyclyl (e.g., a 5- or 6-membered ring);

R^(12a) is H, optionally substituted alkyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, optionally substituted carboxyalkyl (e.g., optionallysubstituted with hydroxy and/or an O-protecting group), optionallysubstituted carboxyalkoxy, optionally substituted carboxyaminoalkyl,optionally substituted carbamoylalkyl, or absent;

R^(12b) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substitutedaminoalkynyl, optionally substituted alkaryl, optionally substitutedheterocyclyl, optionally substituted alkheterocyclyl, optionallysubstituted amino acid, optionally substituted alkoxycarbonylacyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedalkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl,optionally substituted alkoxycarbonylalkynyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl,

wherein the combination of R^(12b) and T^(1′) or the combination ofR^(12b) and R^(12c) can join together to form optionally substitutedheterocyclyl; and

R^(12c) is H, halo, optionally substituted alkyl, optionally substitutedalkoxy, optionally substituted thioalkoxy, optionally substituted amino,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,or optionally substituted aminoalkynyl.

Further exemplary modified uracils include those having Formula(b28)-(b31):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of T¹ and T² is, independently, O (oxo), S (thio), or Se (seleno);

each R^(Vb′) and R^(Vb″) is, independently, H, halo, optionallysubstituted amino acid, optionally substituted alkyl, optionallysubstituted haloalkyl, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted hydroxyalkynyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted alkynyloxy, optionally substituted aminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted acylaminoalkyl (e.g., substituted with an N-protectinggroup, such as any described herein, e.g., trifluoroacetyl), optionallysubstituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g.,optionally substituted with hydroxy and/or an O-protecting group),optionally substituted carboxyalkoxy, optionally substitutedcarboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl) (e.g., R^(Vb′) is optionallysubstituted alkyl, optionally substituted alkenyl, or optionallysubstituted aminoalkyl, e.g., substituted with an N-protecting group,such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl);

R^(12a) is H, optionally substituted alkyl, optionally substitutedcarboxyaminoalkyl, optionally substituted aminoalkyl (e.g., e.g.,substituted with an N-protecting group, such as any described herein,e.g., trifluoroacetyl, or sulfoalkyl), optionally substitutedaminoalkenyl, or optionally substituted aminoalkynyl; and

R^(12b) is H, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted aminoalkyl,optionally substituted aminoalkenyl, optionally substituted aminoalkynyl(e.g., e.g., substituted with an N-protecting group, such as anydescribed herein, e.g., trifluoroacetyl, or sulfoalkyl),

optionally substituted alkoxycarbonylacyl, optionally substitutedalkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy,optionally substituted carboxyalkoxy, optionally substitutedcarboxyalkyl, or optionally substituted carbamoylalkyl.

In particular embodiments, T¹ is O (oxo), and T² is S (thio) or Se(seleno). In other embodiments, T¹ is S (thio), and T² is O (oxo) or Se(seleno). In some embodiments, R^(Vb′) is H, optionally substitutedalkyl, or optionally substituted alkoxy.

In other embodiments, each R^(12a) and R^(12b) is, independently, H,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, or optionally substituted hydroxyalkyl. Inparticular embodiments, R^(12a) is H. In other embodiments, both R^(12a)and R^(12b) are H.

In some embodiments, each R^(Vb′) of R^(12b) is, independently,optionally substituted aminoalkyl (e.g., substituted with anN-protecting group, such as any described herein, e.g., trifluoroacetyl,or sulfoalkyl), optionally substituted aminoalkenyl, optionallysubstituted aminoalkynyl, or optionally substituted acylaminoalkyl(e.g., substituted with an N-protecting group, such as any describedherein, e.g., trifluoroacetyl). In some embodiments, the amino and/oralkyl of the optionally substituted aminoalkyl is substituted with oneor more of optionally substituted alkyl, optionally substituted alkenyl,optionally substituted sulfoalkyl, optionally substituted carboxy (e.g.,substituted with an O-protecting group), optionally substituted hydroxy(e.g., substituted with an O-protecting group), optionally substitutedcarboxyalkyl (e.g., substituted with an O-protecting group), optionallysubstituted alkoxycarbonylalkyl (e.g., substituted with an O-protectinggroup), or N-protecting group. In some embodiments, optionallysubstituted aminoalkyl is substituted with an optionally substitutedsulfoalkyl or optionally substituted alkenyl. In particular embodiments,R^(12a) and R^(Vb″) are both H. In particular embodiments, T¹ is O(oxo), and T² is S (thio) or Se (seleno).

In some embodiments, R^(Vb′) is optionally substitutedalkoxycarbonylalkyl or optionally substituted carbamoylalkyl.

In particular embodiments, the optional substituent for R^(12a),R^(12b), R^(12c), or R^(Va) is a polyethylene glycol group (e.g.,—(CH₂)^(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl); or an amino-polyethylene glycol group (e.g.,—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl).

In some embodiments, B is a modified cytosine. Exemplary modifiedcytosines include compounds of Formula (b10)-(b14):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of T^(3′) and T^(3″) is, independently, H, optionally substitutedalkyl, optionally substituted alkoxy, or optionally substitutedthioalkoxy, or the combination of T^(3′) and T^(3″) join together (e.g.,as in T³) to form O (oxo), S (thio), or Se (seleno);

each V⁴ is, independently, O, S, N(R^(Vc))_(nv), or C(R^(Vc))_(nv),wherein nv is an integer from 0 to 2 and each R^(Vc) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionallysubstituted with any substituent described herein, such as thoseselected from (1)-(21) for alkyl), wherein the combination of R^(13b)and R^(Vc) can be taken together to form optionally substitutedheterocyclyl;

each V⁵ is, independently, N(R^(Vd))_(nv), or C(R^(Vd))_(nv), wherein nvis an integer from 0 to 2 and each R^(Vd) is, independently, H, halo,optionally substituted amino acid, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionallysubstituted with any substituent described herein, such as thoseselected from (1)-(21) for alkyl) (e.g., V⁵ is —CH or N);

each of R^(13a) and R^(13b) is, independently, H, optionally substitutedacyl, optionally substituted acyloxyalkyl, optionally substituted alkyl,or optionally substituted alkoxy, wherein the combination of R^(13b) andR¹⁴ can be taken together to form optionally substituted heterocyclyl;

each R¹⁴ is, independently, H, halo, hydroxy, thiol, optionallysubstituted acyl, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted hydroxyalkyl (e.g., substituted with an O-protecting group),optionally substituted hydroxyalkenyl, optionally substitutedhydroxyalkynyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedacyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H,alkyl, aryl, or phosphoryl), azido, optionally substituted aryl,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, optionally substituted aminoalkyl, optionallysubstituted aminoalkenyl, or optionally substituted aminoalkyl; and

each of R¹⁵ and R¹⁶ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl.

Further exemplary modified cytosines include those having Formula(b32)-(b35):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of T¹ and T³ is, independently, O (oxo), S (thio), or Se (seleno);

each of R^(13a) and R^(13b) is, independently, H, optionally substitutedacyl, optionally substituted acyloxyalkyl, optionally substituted alkyl,or optionally substituted alkoxy, wherein the combination of R^(13b) andR¹⁴ can be taken together to form optionally substituted heterocyclyl;

each R¹⁴ is, independently, H, halo, hydroxy, thiol, optionallysubstituted acyl, optionally substituted amino acid, optionallysubstituted alkyl, optionally substituted haloalkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted hydroxyalkyl (e.g., substituted with an O-protecting group),optionally substituted hydroxyalkenyl, optionally substitutedhydroxyalkynyl, optionally substituted alkoxy, optionally substitutedalkenyloxy, optionally substituted alkynyloxy, optionally substitutedaminoalkoxy, optionally substituted alkoxyalkoxy, optionally substitutedacyloxyalkyl, optionally substituted amino (e.g., —NHR, wherein R is H,alkyl, aryl, or phosphoryl), azido, optionally substituted aryl,optionally substituted heterocyclyl, optionally substitutedalkheterocyclyl, optionally substituted aminoalkyl (e.g., hydroxyalkyl,alkyl, alkenyl, or alkynyl), optionally substituted aminoalkenyl, oroptionally substituted aminoalkynyl; and

each of R¹⁵ and R¹⁶ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl (e.g.,R¹⁵ is H, and R¹⁶ is H or optionally substituted alkyl).

In some embodiments, R¹⁵ is H, and R¹⁶ is H or optionally substitutedalkyl. In particular embodiments, R¹⁴ is H, acyl, or hydroxyalkyl. Insome embodiments, R¹⁴ is halo. In some embodiments, both R¹⁴ and R¹⁵ areH. In some embodiments, both R¹⁵ and R¹⁶ are H. In some embodiments,each of R¹⁴ and R¹⁵ and R¹⁶ is H. In further embodiments, each ofR^(13a) and R^(13b) is independently, H or optionally substituted alkyl.

Further non-limiting examples of modified cytosines include compounds ofFormula (b36):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each R^(13b) is, independently, H, optionally substituted acyl,optionally substituted acyloxyalkyl, optionally substituted alkyl, oroptionally substituted alkoxy, wherein the combination of R^(13b) andR^(14b) can be taken together to form optionally substitutedheterocyclyl;

each R^(14a) and R^(14b) is, independently, H, halo, hydroxy, thiol,optionally substituted acyl, optionally substituted amino acid,optionally substituted alkyl, optionally substituted haloalkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted hydroxyalkyl (e.g., substituted with anO-protecting group), optionally substituted hydroxyalkenyl, optionallysubstituted alkoxy, optionally substituted alkenyloxy, optionallysubstituted alkynyloxy, optionally substituted aminoalkoxy, optionallysubstituted alkoxyalkoxy, optionally substituted acyloxyalkyl,optionally substituted amino (e.g., —NHR, wherein R is H, alkyl, aryl,phosphoryl, optionally substituted aminoalkyl, or optionally substitutedcarboxyaminoalkyl), azido, optionally substituted aryl, optionallysubstituted heterocyclyl, optionally substituted alkheterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,or optionally substituted aminoalkynyl; and

each of R¹⁵ is, independently, H, optionally substituted alkyl,optionally substituted alkenyl, or optionally substituted alkynyl.

In particular embodiments, R^(14b) is an optionally substituted aminoacid (e.g., optionally substituted lysine). In some embodiments, R^(14′)is H.

In some embodiments, B is a modified guanine Exemplary modified guaninesinclude compounds of Formula (b15)-(b17):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of T^(4′), T^(4″), T^(5′), T^(5″), T^(6′), and T^(6″) is,independently, H, optionally substituted alkyl, or optionallysubstituted alkoxy, and wherein the combination of T^(4′) and T^(4″)(e.g., as in T⁴) or the combination of T^(5′) and T^(5″) (e.g., as inT⁵) or the combination of T^(6′) and T^(6″) (e.g., as in T⁶) jointogether form O (oxo), S (thio), or Se (seleno);

each of V⁵ and V⁶ is, independently, O, S, N(R^(Vd))_(nv), orC(R^(Vd))_(nv), wherein nv is an integer from 0 to 2 and each R^(Vd) is,independently, H, halo, thiol, optionally substituted amino acid, cyano,amidine, optionally substituted aminoalkyl, optionally substitutedaminoalkenyl, optionally substituted aminoalkynyl, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted alkoxy, optionallysubstituted alkenyloxy, or optionally substituted alkynyloxy (e.g.,optionally substituted with any substituent described herein, such asthose selected from (1)-(21) for alkyl), optionally substitutedthioalkoxy, or optionally substituted amino; and

each of R¹⁷, R¹⁸, R^(19a), R^(19b), R²¹, R²², R²³, and R²⁴ is,independently, H, halo, thiol, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted thioalkoxy, optionally substituted amino, or optionallysubstituted amino acid.

Exemplary modified guanosines include compounds of Formula (b37)-(b40):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each of T^(4′) is, independently, H, optionally substituted alkyl, oroptionally substituted alkoxy, and each T⁴ is, independently, O (oxo), S(thio), or Se (seleno);

each of R¹⁸, R^(19a), R^(19b), and R²¹ is, independently, H, halo,thiol, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted thioalkoxy,optionally substituted amino, or optionally substituted amino acid.

In some embodiments, R¹⁸ is H or optionally substituted alkyl. Infurther embodiments, T⁴ is oxo. In some embodiments, each of R^(19a) andR^(19b) is, independently, H or optionally substituted alkyl.

In some embodiments, B is a modified adenine. Exemplary modifiedadenines include compounds of Formula (b18)-(b20):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each V⁷ is, independently, O, S, N(R^(Ve))_(nv), or C(R^(Ve))_(nv),wherein nv is an integer from 0 to 2 and each R^(Ve) is, independently,H, halo, optionally substituted amino acid, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted alkoxy, optionally substituted alkenyloxy, oroptionally substituted alkynyloxy (e.g., optionally substituted with anysubstituent described herein, such as those selected from (1)-(21) foralkyl);

each R²⁵ is, independently, H, halo, thiol, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted thioalkoxy, or optionally substituted amino;

each of R^(26a) and R^(26b) is, independently, H, optionally substitutedacyl, optionally substituted amino acid, optionally substitutedcarbamoylalkyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted alkoxy, orpolyethylene glycol group (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl); or an amino-polyethylene glycol group(e.g., —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 isan integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1)is, independently, hydrogen or optionally substituted C₁₋₆ alkyl);

each R²⁷ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted thioalkoxy or optionallysubstituted amino;

each R²⁸ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, or optionally substituted alkynyl; and

each R²⁹ is, independently, H, optionally substituted acyl, optionallysubstituted amino acid, optionally substituted carbamoylalkyl,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted hydroxyalkyl, optionallysubstituted hydroxyalkenyl, optionally substituted alkoxy, or optionallysubstituted amino.

Exemplary modified adenines include compounds of Formula (b41)-(b43):

or a pharmaceutically acceptable salt or stereoisomer thereof,

wherein

each R²⁵ is, independently, H, halo, thiol, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted thioalkoxy, or optionally substituted amino;

each of R^(26a) and R^(26b) is, independently, H, optionally substitutedacyl, optionally substituted amino acid, optionally substitutedcarbamoylalkyl, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substitutedhydroxyalkyl, optionally substituted hydroxyalkenyl, optionallysubstituted hydroxyalkynyl, optionally substituted alkoxy, orpolyethylene glycol group (e.g., —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′,wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g.,from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10),and R′ is H or C₁₋₂₀ alkyl); or an amino-polyethylene glycol group(e.g., —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 isan integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4,from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1)is, independently, hydrogen or optionally substituted C₁₋₆ alkyl); and

each R²⁷ is, independently, H, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted alkoxy, optionally substituted thioalkoxy, or optionallysubstituted amino.

In some embodiments, R^(26a) is H, and R^(26b) is optionally substitutedalkyl. In some embodiments, each of R^(26a) and R^(26b) is,independently, optionally substituted alkyl. In particular embodiments,R²⁷ is optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy. In other embodiments, R²⁵ isoptionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted thioalkoxy.

In particular embodiments, the optional substituent for R^(26a),R^(26b), or R²⁹ is a polyethylene glycol group (e.g.,—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl); or an amino-polyethylene glycol group (e.g.,—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl).

In some embodiments, B may have Formula (b21):

wherein X¹² is, independently, O, S, optionally substituted alkylene(e.g., methylene), or optionally substituted heteroalkylene, xa is aninteger from 0 to 3, and R^(12a) and T² are as described herein.

In some embodiments, B may have Formula (b22):

wherein R^(10′) is, independently, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, optionally substituted alkoxy,optionally substituted alkoxycarbonylalkyl, optionally substitutedalkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl, or optionallysubstituted carbamoylalkyl, and R¹¹, R^(12a), T¹, and T² are asdescribed herein.

In some embodiments, B may have Formula (b23):

wherein R¹⁰ is optionally substituted heterocyclyl (e.g., optionallysubstituted furyl, optionally substituted thienyl, or optionallysubstituted pyrrolyl), optionally substituted aryl (e.g., optionallysubstituted phenyl or optionally substituted naphthyl), or anysubstituent described herein (e.g., for)R¹⁰; and wherein R¹¹ (e.g., H orany substituent described herein), R^(12a) (e.g., H or any substituentdescribed herein), T¹ (e.g., oxo or any substituent described herein),and T² (e.g., oxo or any substituent described herein) are as describedherein.

In some embodiments, B may have Formula (b24):

wherein R^(14′) is, independently, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted aryl, optionally substituted heterocyclyl,optionally substituted alkaryl, optionally substituted alkheterocyclyl,optionally substituted aminoalkyl, optionally substituted aminoalkenyl,optionally substituted aminoalkynyl, optionally substituted alkoxy,optionally substituted alkoxycarbonylalkenyl, optionally substitutedalkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkyl,optionally substituted alkoxycarbonylalkoxy, optionally substitutedcarboxyalkoxy, optionally substituted carboxyalkyl, or optionallysubstituted carbamoylalkyl, and R^(13a), R^(13b), R¹⁵, and T³ are asdescribed herein.

In some embodiments, B may have Formula (b25):

wherein R^(14′) is optionally substituted heterocyclyl (e.g., optionallysubstituted furyl, optionally substitued thienyl, or optionallysubstitued pyrrolyl), optionally substituted aryl (e.g., optionallysubstituted phenyl or optionally substituted naphthyl), or anysubstituent described herein (e.g., for R¹⁴ or R^(14′)); and whereinR^(13a) (e.g., H or any substituent described herein), R^(13b) (e.g., Hor any substituent described herein), R¹⁵ (e.g., H or any substituentdescribed herein), and T³ (e.g., oxo or any substituent describedherein) are as described herein.

In some embodiments, B is a nucleobase selected from the groupconsisting of cytosine, guanine, adenine, and uracil. In someembodiments, B may be:

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm⁵s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e.,having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine (m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um),5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴2 Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m′A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶2Am), 1,2′-O-dimethyl-adenosine (m′Am), 2′-O-ribosyladenosine (phosphate)(Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine,2′-F-ara-adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, andN6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanineExemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodifiedhydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m²2 G), N2,7-dimethyl-guanosine (m^(2,7)G),N2, N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m²2 Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m¹Im), and 2′-O-ribosylguanosine(phosphate) (Gr(p)).

The nucleobase of the nucleotide can be independently selected from apurine, a pyrimidine, a purine or pyrimidine analog. For example, thenucleobase can each be independently selected from adenine, cytosine,guanine, uracil, or hypoxanthine. In another embodiment, the nucleobasecan also include, for example, naturally-occurring and syntheticderivatives of a base, including pyrazolo[3,4-d]pyrimidines,5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol,8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines,5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituteduracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanineand 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine,deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine,imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines,imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones,1,2,4-triazine, pyridazine; and 1,3,5 triazine. When the nucleotides aredepicted using the shorthand A, G, C, T or U, each letter refers to therepresentative base and/or derivatives thereof, e.g., A includes adenineor adenine analogs, e.g., 7-deaza adenine).

Modifications on the Internucleoside Linkage

The modified nucleotides, which may be incorporated into apolynucleotide, primary construct, or mmRNA molecule, can be modified onthe internucleoside linkage (e.g., phosphate backbone). Herein, in thecontext of the polynucleotide backbone, the phrases “phosphate” and“phosphodiester” are used interchangeably. Backbone phosphate groups canbe modified by replacing one or more of the oxygen atoms with adifferent substituent. Further, the modified nucleosides and nucleotidescan include the wholesale replacement of an unmodified phosphate moietywith another internucleoside linkage as described herein. Examples ofmodified phosphate groups include, but are not limited to,phosphorothioate, phosphoroselenates, boranophosphates, boranophosphateesters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates,alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioateshave both non-linking oxygens replaced by sulfur. The phosphate linkercan also be modified by the replacement of a linking oxygen withnitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates),and carbon (bridged methylene-phosphonates).

The α-thio substituted phosphate moiety is provided to confer stabilityto RNA and DNA polymers through the unnatural phosphorothioate backbonelinkages. Phosphorothioate DNA and RNA have increased nucleaseresistance and subsequently a longer half-life in a cellularenvironment. Phosphorothioate linked polynucleotides, primaryconstructs, or mmRNA molecules are expected to also reduce the innateimmune response through weaker binding/activation of cellular innateimmune molecules.

In specific embodiments, a modified nucleoside includes analpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine,5′-O-(1-thiophosphate)-cytidine (α-thio-cytidine),5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or5′-O-(1-thiophosphate)-pseudouridine).

Other internucleoside linkages that may be employed according to thepresent invention, including internucleoside linkages which do notcontain a phosphorous atom, are described herein below.

Major Groove Interacting Partners

As described herein, the phrase “major groove interacting partner”refers to RNA recognition receptors that detect and respond to RNAligands through interactions, e.g. binding, with the major groove faceof a nucleotide or nucleic acid. As such, RNA ligands comprisingmodified nucleotides or nucleic acids such as the polynucleotide,primary construct or mmRNA as described herein decrease interactionswith major groove binding partners, and therefore decrease an innateimmune response.

Example major groove interacting, e.g. binding, partners include, butare not limited to the following nucleases and helicases. Withinmembranes, TLRs (Toll-like Receptors) 3, 7, and 8 can respond to single-and double-stranded RNAs. Within the cytoplasm, members of thesuperfamily 2 class of DEX(D/H) helicases and ATPases can sense RNAs toinitiate antiviral responses. These helicases include the RIG-I(retinoic acid-inducible gene I) and MDA5 (melanomadifferentiation-associated gene 5). Other examples include laboratory ofgenetics and physiology 2 (LGP2), HIN-200 domain containing proteins, orHelicase-domain containing proteins.

Combinations of Modified Sugars, Nucleobases, and InternucleosideLinkages

The polynucleotides, primary constructs, and mmRNA of the invention caninclude a combination of modifications to the sugar, the nucleobase,and/or the internucleoside linkage. These combinations can include anyone or more modifications described herein. For examples, any of thenucleotides described herein in Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If),(IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2),(IVa)-(IV1), and (IXa)-(IXr) can be combined with any of the nucleobasesdescribed herein (e.g., in Formulas (b1)-(b43) or any other describedherein).

Synthesis of Polypeptides, Primary Constructs, and mmRNA Molecules

The polypeptides, primary constructs, and mmRNA molecules for use inaccordance with the invention may be prepared according to any usefultechnique, as described herein. The modified nucleosides and nucleotidesused in the synthesis of polynucleotides, primary constructs, and mmRNAmolecules disclosed herein can be prepared from readily availablestarting materials using the following general methods and procedures.Where typical or preferred process conditions (e.g., reactiontemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are provided, a skilled artisan would be able to optimize anddevelop additional process conditions. Optimum reaction conditions mayvary with the particular reactants or solvent used, but such conditionscan be determined by one skilled in the art by routine optimizationprocedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry(e.g., UV-visible), or mass spectrometry, or by chromatography such ashigh performance liquid chromatography (HPLC) or thin layerchromatography.

Preparation of polypeptides, primary constructs, and mmRNA molecules ofthe present invention can involve the protection and deprotection ofvarious chemical groups. The need for protection and deprotection, andthe selection of appropriate protecting groups can be readily determinedby one skilled in the art. The chemistry of protecting groups can befound, for example, in Greene, et al., Protective Groups in OrganicSynthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein byreference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents, which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of modified nucleosides and nucleotidescan be carried out by any of numerous methods known in the art. Anexample method includes fractional recrystallization using a “chiralresolving acid” which is an optically active, salt-forming organic acid.Suitable resolving agents for fractional recrystallization methods are,for example, optically active acids, such as the D and L forms oftartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelicacid, malic acid, lactic acid or the various optically activecamphorsulfonic acids. Resolution of racemic mixtures can also becarried out by elution on a column packed with an optically activeresolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elutionsolvent composition can be determined by one skilled in the art.

Modified nucleosides and nucleotides (e.g., building block molecules)can be prepared according to the synthetic methods described in Ogata etal., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568(1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each ofwhich are incorporated by reference in their entirety.

The polypeptides, primary constructs, and mmRNA of the invention may ormay not be uniformly modified along the entire length of the molecule.For example, one or more or all types of nucleotide (e.g., purine orpyrimidine, or any one or more or all of A, G, U, C) may or may not beuniformly modified in a polynucleotide of the invention, or in a givenpredetermined sequence region thereof (e.g. one or more of the sequenceregions represented in FIG. 1). In some embodiments, all nucleotides Xin a polynucleotide of the invention (or in a given sequence regionthereof) are modified, wherein X may any one of nucleotides A, G, U, C,or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U,A+G+C, G+U+C or A+G+C.

Different sugar modifications, nucleotide modifications, and/orinternucleoside linkages (e.g., backbone structures) may exist atvarious positions in the polynucleotide, primary construct, or mmRNA.One of ordinary skill in the art will appreciate that the nucleotideanalogs or other modification(s) may be located at any position(s) of apolynucleotide, primary construct, or mmRNA such that the function ofthe polynucleotide, primary construct, or mmRNA is not substantiallydecreased. A modification may also be a 5′ or 3′ terminal modification.The polynucleotide, primary construct, or mmRNA may contain from about1% to about 100% modified nucleotides (either in relation to overallnucleotide content, or in relation to one or more types of nucleotide,i.e. any one or more of A, G, U or C) or any intervening percentage(e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%,from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10%to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100%).

In some embodiments, the polynucleotide, primary construct, or mmRNAincludes a modified pyrimidine (e.g., a modified uracil/uridine/U ormodified cytosine/cytidine/C). In some embodiments, the uracil oruridine (generally: U) in the polynucleotide, primary construct, ormmRNA molecule may be replaced with from about 1% to about 100% of amodified uracil or modified uridine (e.g., from 1% to 20%, from 1% to25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100% of a modified uracil or modifieduridine). The modified uracil or uridine can be replaced by a compoundhaving a single unique structure or by a plurality of compounds havingdifferent structures (e.g., 2, 3, 4 or more unique structures, asdescribed herein). In some embodiments, the cytosine or cytidine(generally: C) in the polynucleotide, primary construct, or mmRNAmolecule may be replaced with from about 1% to about 100% of a modifiedcytosine or modified cytidine (e.g., from 1% to 20%, from 1% to 25%,from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1%to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%,from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%,from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50%to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to100%, and from 95% to 100% of a modified cytosine or modified cytidine).The modified cytosine or cytidine can be replaced by a compound having asingle unique structure or by a plurality of compounds having differentstructures (e.g., 2, 3, 4 or more unique structures, as describedherein).

In some embodiments, the present disclosure provides methods ofsynthesizing a polynucleotide, primary construct, or mmRNA (e.g., thefirst region, first flanking region, or second flanking region)including n number of linked nucleosides having Formula (Ia-1):

comprising:

a) reacting a nucleotide of Formula (IV-1):

with a phosphoramidite compound of Formula (V-1):

wherein Y⁹ is H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino,thiol, optionally substituted amino acid, or a peptide (e.g., includingfrom 2 to 12 amino acids); and each P¹, P², and P³ is, independently, asuitable protecting group; and

denotes a solid support;

to provide a polynucleotide, primary construct, or mmRNA of Formula(VI-1):

and

b) oxidizing or sulfurizing the polynucleotide, primary construct, ormmRNA of Formula (V) to yield a polynucleotide, primary construct, ormmRNA of Formula (VII-1):

and

c) removing the protecting groups to yield the polynucleotide, primaryconstruct, or mmRNA of Formula (Ia).

In some embodiments, steps a) and b) are repeated from 1 to about 10,000times. In some embodiments, the methods further comprise a nucleotide(e.g., mmRNA molecule) selected from the group consisting of A, C, G andU adenosine, cytosine, guanosine, and uracil. In some embodiments, thenucleobase may be a pyrimidine or derivative thereof. In someembodiments, the polynucleotide, primary construct, or mmRNA istranslatable.

Other components of polynucleotides, primary constructs, and mmRNA areoptional, and are beneficial in some embodiments. For example, a 5′untranslated region (UTR) and/or a 3′UTR are provided, wherein either orboth may independently contain one or more different nucleotidemodifications. In such embodiments, nucleotide modifications may also bepresent in the translatable region. Also provided are polynucleotides,primary constructs, and mmRNA containing a Kozak sequence. Exemplarysyntheses of modified nucleotides, which are incorporated into amodified nucleic acid or mmRNA, e.g., RNA or mRNA, are provided below inScheme 1 through Scheme 11. Scheme 1 provides a general method forphosphorylation of nucleosides, including modified nucleosides.

Various protecting groups may be used to control the reaction. Forexample, Scheme 2 provides the use of multiple protecting anddeprotecting steps to promote phosphorylation at the 5′ position of thesugar, rather than the 2′ and 3′ hydroxyl groups.

Modified nucleotides can be synthesized in any useful manner. Schemes 3,4, and 7 provide exemplary methods for synthesizing modified nucleotideshaving a modified purine nucleobase; and Schemes 5 and 6 provideexemplary methods for synthesizing modified nucleotides having amodified pseudouridine or pseudoisocytidine, respectively.

Schemes 8 and 9 provide exemplary syntheses of modified nucleotides.Scheme 10 provides a non-limiting biocatalytic method for producingnucleotides.

Scheme 11 provides an exemplary synthesis of a modified uracil, wherethe N1 position is modified with R^(12b), as provided elsewhere, and the5′-position of ribose is phosphorylated. T¹, T², R^(12a), R^(12b), and rare as provided herein. This synthesis, as well as optimized versionsthereof, can be used to modify other pyrimidine nucleobases and purinenucleobases (see e.g., Formulas (b1)-(b43)) and/or to install one ormore phosphate groups (e.g., at the 5′ position of the sugar). Thisalkylating reaction can also be used to include one or more optionallysubstituted alkyl group at any reactive group (e.g., amino group) in anynucleobase described herein (e.g., the amino groups in the Watson-Crickbase-pairing face for cytosine, uracil, adenine, and guanine)

Combinations of Nucleotides in mmRNA

Further examples of modified nucleotides and modified nucleotidecombinations are provided below in Table 9. These combinations ofmodified nucleotides can be used to form the polypeptides, primaryconstructs, or mmRNA of the invention. Unless otherwise noted, themodified nucleotides may be completely substituted for the naturalnucleotides of the modified nucleic acids or mmRNA of the invention. Asa non-limiting example, the natural nucleotide uridine may besubstituted with a modified nucleoside described herein. In anothernon-limiting example, the natural nucleotide uridine may be partiallysubstituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%)with at least one of the modified nucleoside disclosed herein.

TABLE 9 Modified Nucleotide Modified Nucleotide Combinationα-thio-cytidine α-thio-cytidine/5-iodo-uridineα-thio-cytidine/N1-methyl-pseudo-uridine α-thio-cytidine/α-thio-uridineα-thio-cytidine/5-methyl-uridine α-thio-cytidine/pseudo-uridine about50% of the cytosines are α-thio-cytidine pseudoisocytidinepseudoisocytidine/5-iodo-uridinepseudoisocytidine/N1-methyl-pseudouridinepseudoisocytidine/α-thio-uridine pseudoisocytidine/5-methyl-uridinepseudoisocytidine/pseudouridine about 25% of cytosines arepseudoisocytidine pseudoisocytidine/about 50% of uridines are N1-methyl-pseudouridine and about 50% of uridines are pseudouridinepseudoisocytidine/about 25% of uridines are N1-methyl-pseudouridine andabout 25% of uridines are pseudouridine pyrrolo-cytidinepyrrolo-cytidine/5-iodo-uridine pyrrolo-cytidine/N1-methyl-pseudouridinepyrrolo-cytidine/α-thio-uridine pyrrolo-cytidine/5-methyl-uridinepyrrolo-cytidine/pseudouridine about 50% of the cytosines arepyrrolo-cytidine 5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine5-methyl-cytidine/N1-methyl-pseudouridine5-methyl-cytidine/α-thio-uridine 5-methyl-cytidine/5-methyl-uridine5-methyl-cytidine/pseudouridine about 25% of cytosines are5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridinesare 2-thio-uridine about 50% of uridines are 5-methyl-cytidine/about 50%of uridines are 2-thio-uridine N4-acetyl-cytidineN4-acetyl-cytidine/5-iodo-uridineN4-acetyl-cytidine/N1-methyl-pseudouridineN4-acetyl-cytidine/α-thio-uridine N4-acetyl-cytidine/5-methyl-uridineN4-acetyl-cytidine/pseudouridine about 50% of cytosines areN4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidineN4-acetyl-cytidine/5-methoxy-uridine N4-acetyl-cytidine/5-bromo-uridineN4-acetyl-cytidine/2-thio-uridine about 50% of cytosines areN4-acetyl-cytidine/about 50% of uridines are 2-thio-uridine

Further examples of modified nucleotide combinations are provided belowin Table 10. These combinations of modified nucleotides can be used toform the polypeptides, primary constructs, or mmRNA of the invention.

TABLE 10 Modified Nucleotide Modified Nucleotide Combination modifiedcytidine having modified cytidine with (b10)/pseudouridine one or morenucleobases modified cytidine with (b10)/N1-methyl-pseudouridine ofFormula (b10) modified cytidine with (b10)/5-methoxy-uridine modifiedcytidine with (b10)/5-methyl-uridine modified cytidine with(b10)/5-bromo-uridine modified cytidine with (b10)/2-thio-uridine about50% of cytidine substituted with modified cytidine (b10)/ about 50% ofuridines are 2-thio-uridine modified cytidine having modified cytidinewith (b32)/pseudouridine one or more nucleobases modified cytidine with(b32)/N1-methyl-pseudouridine of Formula (b32) modified cytidine with(b32)/5-methoxy-uridine modified cytidine with (b32)/5-methyl-uridinemodified cytidine with (b32)/5-bromo-uridine modified cytidine with(b32)/2-thio-uridine about 50% of cytidine substituted with modifiedcytidine (b32)/ about 50% of uridines are 2-thio-uridine modifieduridine having modified uridine with (b1)/N4-acetyl-cytidine one or morenucleobases modified uridine with (b1)/5-methyl-cytidine of Formula (b1)modified uridine having modified uridine with (b8)/N4-acetyl-cytidineone or more nucleobases modified uridine with (b8)/5-methyl-cytidine ofFormula (b8) modified uridine having modified uridine with(b28)/N4-acetyl-cytidine one or more nucleobases modified uridine with(b28)/5-methyl-cytidine of Formula (b28) modified uridine havingmodified uridine with (b29)/N4-acetyl-cytidine one or more nucleobasesmodified uridine with (b29)/5-methyl-cytidine of Formula (b29) modifieduridine having modified uridine with (b30)/N4-acetyl-cytidine one ormore nucleobases modified uridine with (b30)/5-methyl-cytidine ofFormula (b30)

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14) (e.g., at least about 30%, at leastabout 35%, at least about 40%, at least about 45%, at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the uracils are replaced by acompound of Formula (b1)-(b9) (e.g., at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%).

In some embodiments, at least 25% of the cytosines are replaced by acompound of Formula (b10)-(b14), and at least 25% of the uracils arereplaced by a compound of Formula (b1)-(b9) (e.g., at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or about 100%).

IV. PHARMACEUTICAL COMPOSITIONS Formulation, Administration, Deliveryand Dosing

The present invention provides polynucleotides, primary constructs andmmRNA compositions and complexes in combination with one or morepharmaceutically acceptable excipients. Pharmaceutical compositions mayoptionally comprise one or more additional active substances, e.g.therapeutically and/or prophylactically active substances. Generalconsiderations in the formulation and/or manufacture of pharmaceuticalagents may be found, for example, in Remington: The Science and Practiceof Pharmacy 21^(st) ed., Lippincott Williams & Wilkins, 2005(incorporated herein by reference).

In some embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to polynucleotides, primaryconstructs and mmRNA to be delivered as described herein.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to any other animal, e.g., to non-human animals, e.g.non-human mammals. Modification of pharmaceutical compositions suitablefor administration to humans in order to render the compositionssuitable for administration to various animals is well understood, andthe ordinarily skilled veterinary pharmacologist can design and/orperform such modification with merely ordinary, if any, experimentation.Subjects to which administration of the pharmaceutical compositions iscontemplated include, but are not limited to, humans and/or otherprimates; mammals, including commercially relevant mammals such ascattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/orbirds, including commercially relevant birds such as poultry, chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, dividing, shaping and/or packaging the product into a desiredsingle- or multi-dose unit.

A pharmaceutical composition in accordance with the invention may beprepared, packaged, and/or sold in bulk, as a single unit dose, and/oras a plurality of single unit doses. As used herein, a “unit dose” isdiscrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the invention will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

Formulations

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using one or more excipients to: (1) increase stability; (2)increase cell transfection; (3) permit the sustained or delayed release(e.g., from a depot formulation of the polynucleotide, primaryconstruct, or mmRNA); (4) alter the biodistribution (e.g., target thepolynucleotide, primary construct, or mmRNA to specific tissues or celltypes); (5) increase the translation of encoded protein in vivo; and/or(6) alter the release profile of encoded protein in vivo. In addition totraditional excipients such as any and all solvents, dispersion media,diluents, or other liquid vehicles, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, excipients of the present invention can include,without limitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with polynucleotide, primary construct, or mmRNA (e.g., fortransplantation into a subject), hyaluronidase, nanoparticle mimicsandcombinations thereof. Accordingly, the formulations of the invention caninclude one or more excipients, each in an amount that togetherincreases the stability of the polynucleotide, primary construct, ormmRNA, increases cell transfection by the polynucleotide, primaryconstruct, or mmRNA, increases the expression of polynucleotide, primaryconstruct, or mmRNA encoded protein, and/or alters the release profileof polynucleotide, primary construct, or mmRNA encoded proteins.Further, the primary construct and mmRNA of the present invention may beformulated using self-assembled nucleic acid nanoparticles.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” refers to a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient may generally be equal to the dosage of theactive ingredient which would be administered to a subject and/or aconvenient fraction of such a dosage including, but not limited to,one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure mayvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered. For example, the composition maycomprise between 0.1% and 99% (w/w) of the active ingredient.

In some embodiments, the formulations described herein may contain atleast one mmRNA. As a non-limiting example, the formulations may contain1, 2, 3, 4 or 5 mmRNA. In one embodiment the formulation may containmodified mRNA encoding proteins selected from categories such as, butnot limited to, human proteins, veterinary proteins, bacterial proteins,biological proteins, antibodies, immunogenic proteins, therapeuticpeptides and proteins, secreted proteins, plasma membrane proteins,cytoplasmic and cytoskeletal proteins, intrancellular membrane boundproteins, nuclear proteins, proteins associated with human diseaseand/or proteins associated with non-human diseases. In one embodiment,the formulation contains at least three modified mRNA encoding proteins.In one embodiment, the formulation contains at least five modified mRNAencoding proteins.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes, but is notlimited to, any and all solvents, dispersion media, diluents, or otherliquid vehicles, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, andthe like, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro,Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference). The use of a conventional excipient medium may becontemplated within the scope of the present disclosure, except insofaras any conventional excipient medium may be incompatible with asubstance or its derivatives, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition.

In some embodiments, the particle size of the lipid nanoparticle may beincreased and/or decreased. The change in particle size may be able tohelp counter biological reaction such as, but not limited to,inflammation or may increase the biological effect of the modified mRNAdelivered to mammals.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, surface active agents and/or emulsifiers, preservatives,buffering agents, lubricating agents, and/or oils. Such excipients mayoptionally be included in the pharmaceutical formulations of theinvention.

Lipidoids

The synthesis of lipidoids has been extensively described andformulations containing these compounds are particularly suited fordelivery of polynucleotides, primary constructs or mmRNA (see Mahon etal., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med.2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., ProcNatl Acad Sci USA. 2011 108:12996-3001; all of which are incorporatedherein in their entireties).

While these lipidoids have been used to effectively deliver doublestranded small interfering RNA molecules in rodents and non-humanprimates (see Akinc et al., Nat Biotechnol. 2008 26:561-569;Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920;Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad SciUSA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 201129:1005-1010; all of which is incorporated herein in their entirety),the present disclosure describes their formulation and use in deliveringsingle stranded polynucleotides, primary constructs, or mmRNA.Complexes, micelles, liposomes or particles can be prepared containingthese lipidoids and therefore, can result in an effective delivery ofthe polynucleotide, primary construct, or mmRNA, as judged by theproduction of an encoded protein, following the injection of a lipidoidformulation via localized and/or systemic routes of administration.Lipidoid complexes of polynucleotides, primary constructs, or mmRNA canbe administered by various means including, but not limited to,intravenous, intramuscular, or subcutaneous routes.

In vivo delivery of nucleic acids may be affected by many parameters,including, but not limited to, the formulation composition, nature ofparticle PEGylation, degree of loading, oligonucleotide to lipid ratio,and biophysical parameters such as particle size (Akinc et al., MolTher. 2009 17:872-879; herein incorporated by reference in itsentirety). As an example, small changes in the anchor chain length ofpoly(ethylene glycol) (PEG) lipids may result in significant effects onin vivo efficacy. Formulations with the different lipidoids, including,but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry,401:61 (2010)), C12-200 (including derivatives and variants), and MD1,can be tested for in vivo activity.

The lipidoid referred to herein as “98N12-5” is disclosed by Akinc etal., Mol Ther. 2009 17:872-879 and is incorporated by reference in itsentirety.

The lipidoid referred to herein as “C12-200” is disclosed by Love etal., Proc Natl Acad Sci USA. 2010 107:1864-1869 and Liu and Huang,Molecular Therapy. 2010 669-670; both of which are herein incorporatedby reference in their entirety. The lipidoid formulations can includeparticles comprising either 3 or 4 or more components in addition topolynucleotide, primary construct, or mmRNA. As an example, formulationswith certain lipidoids, include, but are not limited to, 98N12-5 and maycontain 42% lipidoid, 48% cholesterol and 10% PEG (C14 alkyl chainlength). As another example, formulations with certain lipidoids,include, but are not limited to, C12-200 and may contain 50% lipidoid,10% disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.

In one embodiment, a polynucleotide, primary construct, or mmRNAformulated with a lipidoid for systemic intravenous administration cantarget the liver. For example, a final optimized intravenous formulationusing polynucleotide, primary construct, or mmRNA, and comprising alipid molar composition of 42% 98N12-5, 48% cholesterol, and 10%PEG-lipid with a final weight ratio of about 7.5 to 1 total lipid topolynucleotide, primary construct, or mmRNA, and a C14 alkyl chainlength on the PEG lipid, with a mean particle size of roughly 50-60 nm,can result in the distribution of the formulation to be greater than 90%to the liver. (see, Akinc et al., Mol Ther. 2009 17:872-879; hereinincorporated in its entirety). In another example, an intravenousformulation using a C12-200 (see U.S. provisional application 61/175,770and published international application WO2010129709, hereinincorporated by reference in their entirety) lipidoid may have a molarratio of 50/10/38.5/1.5 of C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG, with a weight ratio of 7 to 1 total lipidto polynucleotide, primary construct, or mmRNA, and a mean particle sizeof 80 nm may be effective to deliver polynucleotide, primary construct,or mmRNA to hepatocytes (see, Love et al., Proc Natl Acad Sci USA. 2010107:1864-1869 herein incorporated by reference). In another embodiment,an MD1 lipidoid-containing formulation may be used to effectivelydeliver polynucleotide, primary construct, or mmRNA to hepatocytes invivo. The characteristics of optimized lipidoid formulations forintramuscular or subcutaneous routes may vary significantly depending onthe target cell type and the ability of formulations to diffuse throughthe extracellular matrix into the blood stream. While a particle size ofless than 150 nm may be desired for effective hepatocyte delivery due tothe size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 200917:872-879 herein incorporated by reference), use of alipidoid-formulated polynucleotide, primary construct, or mmRNA todeliver the formulation to other cells types including, but not limitedto, endothelial cells, myeloid cells, and muscle cells may not besimilarly size-limited. Use of lipidoid formulations to deliver siRNA invivo to other non-hepatocyte cells such as myeloid cells and endotheliumhas been reported (see Akinc et al., Nat Biotechnol. 2008 26:561-569;Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; Cho et al. Adv.Funct. Mater. 2009 19:3112-3118; 8^(th) International Judah FolkmanConference, Cambridge, Mass. Oct. 8-9, 2010 herein incorporated byreference in its entirety). Effective delivery to myeloid cells, such asmonocytes, lipidoid formulations may have a similar component molarratio. Different ratios of lipidoids and other components including, butnot limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG,may be used to optimize the formulation of the polynucleotide, primaryconstruct, or mmRNA for delivery to different cell types including, butnot limited to, hepatocytes, myeloid cells, muscle cells, etc. Forexample, the component molar ratio may include, but is not limited to,50% C12-200, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and%1.5 PEG-DMG (see Leuschner et al., Nat Biotechnol 2011 29:1005-1010;herein incorporated by reference in its entirety). The use of lipidoidformulations for the localized delivery of nucleic acids to cells (suchas, but not limited to, adipose cells and muscle cells) via eithersubcutaneous or intramuscular delivery, may not require all of theformulation components desired for systemic delivery, and as such maycomprise only the lipidoid and the polynucleotide, primary construct, ormmRNA.

Combinations of different lipidoids may be used to improve the efficacyof polynucleotide, primary construct, or mmRNA directed proteinproduction as the lipidoids may be able to increase cell transfection bythe polynucleotide, primary construct, or mmRNA; and/or increase thetranslation of encoded protein (see Whitehead et al., Mol. Ther. 2011,19:1688-1694, herein incorporated by reference in its entirety).

Liposomes, Lipoplexes, and Lipid Nanoparticles

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using one or more liposomes, lipoplexes, or lipidnanoparticles. In one embodiment, pharmaceutical compositions ofpolynucleotide, primary construct, or mmRNA include liposomes. Liposomesare artificially-prepared vesicles which may primarily be composed of alipid bilayer and may be used as a delivery vehicle for theadministration of nutrients and pharmaceutical formulations. Liposomescan be of different sizes such as, but not limited to, a multilamellarvesicle (MLV) which may be hundreds of nanometers in diameter and maycontain a series of concentric bilayers separated by narrow aqueouscompartments, a small unicellular vesicle (SUV) which may be smallerthan 50 nm in diameter, and a large unilamellar vesicle (LUV) which maybe between 50 and 500 nm in diameter. Liposome design may include, butis not limited to, opsonins or ligands in order to improve theattachment of liposomes to unhealthy tissue or to activate events suchas, but not limited to, endocytosis. Liposomes may contain a low or ahigh pH in order to improve the delivery of the pharmaceuticalformulations.

The formation of liposomes may depend on the physicochemicalcharacteristics such as, but not limited to, the pharmaceuticalformulation entrapped and the liposomal ingredients, the nature of themedium in which the lipid vesicles are dispersed, the effectiveconcentration of the entrapped substance and its potential toxicity, anyadditional processes involved during the application and/or delivery ofthe vesicles, the optimization size, polydispersity and the shelf-lifeof the vesicles for the intended application, and the batch-to-batchreproducibility and possibility of large-scale production of safe andefficient liposomal products.

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2liposomes from Marina Biotech (Bothell, Wash.),1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA),and MC3 (US20100324120; herein incorporated by reference in itsentirety) and liposomes which may deliver small molecule drugs such as,but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

In one embodiment, pharmaceutical compositions described herein mayinclude, without limitation, liposomes such as those formed from thesynthesis of stabilized plasmid-lipid particles (SPLP) or stabilizednucleic acid lipid particle (SNALP) that have been previously describedand shown to be suitable for oligonucleotide delivery in vitro and invivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. GeneTherapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372;Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al.,Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J ClinInvest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132;all of which are incorporated herein in their entireties.) The originalmanufacture method by Wheeler et al. was a detergent dialysis method,which was later improved by Jeffs et al. and is referred to as thespontaneous vesicle formation method. The liposome formulations arecomposed of 3 to 4 lipid components in addition to the polynucleotide,primary construct, or mmRNA. As an example a liposome can contain, butis not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline(DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane(DODMA), as described by Jeffs et al. As another example, certainliposome formulations may contain, but are not limited to, 48%cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where thecationic lipid can be 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA),DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA),as described by Heyes et al.

In one embodiment, pharmaceutical compositions may include liposomeswhich may be formed to deliver mmRNA which may encode at least oneimmunogen. The mmRNA may be encapsulated by the liposome and/or it maybe contained in an aqueous core which may then be encapsulated by theliposome (see International Pub. Nos. WO2012031046, WO2012031043,WO2012030901 and WO2012006378 herein incorporated by reference in theirentireties). In another embodiment, the mmRNA which may encode animmunogen may be formulated in a cationic oil-in-water emulsion wherethe emulsion particle comprises an oil core and a cationic lipid whichcan interact with the mmRNA anchoring the molecule to the emulsionparticle (see International Pub. No. WO2012006380). In yet anotherembodiment, the lipid formulation may include at least cationic lipid, alipid which may enhance transfection and a least one lipid whichcontains a hydrophilic head group linked to a lipid moiety(International Pub. No. WO2011076807 and U.S. Pub. No. 20110200582;herein incorporated by reference in their entireties). In anotherembodiment, the polynucleotides, primary constructs and/or mmRNAencoding an immunogen may be formulated in a lipid vesicle which mayhave crosslinks between functionalized lipid bilayers (see U.S. Pub. No.20120177724, herein incorporated by reference in its entirety).

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be formulated in a lipid vesicle which may have crosslinks betweenfunctionalized lipid bilayers.

In one embodiment, the polynucleotides, primary constructs and/or mmRNAmay be formulated in a lipid-polycation complex. The formation of thelipid-polycation complex may be accomplished by methods known in the artand/or as described in U.S. Pub. No. 20120178702, herein incorporated byreference in its entirety. As a non-limiting example, the polycation mayinclude a cationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine. In another embodiment,the polynucleotides, primary constructs and/or mmRNA may be formulatedin a lipid-polycation complex which may further include a neutral lipidsuch as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

The liposome formulation may be influenced by, but not limited to, theselection of the cationic lipid component, the degree of cationic lipidsaturation, the nature of the PEGylation, ratio of all components andbiophysical parameters such as size. In one example by Semple et al.(Semple et al. Nature Biotech. 2010 28:172-176), the liposomeformulation was composed of 57.1% cationic lipid, 7.1%dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.As another example, changing the composition of the cationic lipid couldmore effectively deliver siRNA to various antigen presenting cells(Basha et al. Mol Ther. 2011 19:2186-2200; herein incorporated byreference in its entirety).

In some embodiments, the ratio of PEG in the LNP formulations may beincreased or decreased and/or the carbon chain length of the PEG lipidmay be modified from C14 to C18 to alter the pharmacokinetics and/orbiodistribution of the LNP formulations. As a non-limiting example, LNPformulations may contain 1-5% of the lipid molar ratio of PEG-c-DOMG ascompared to the cationic lipid, DSPC and cholesterol. In anotherembodiment the PEG-c-DOMG may be replaced with a PEG lipid such as, butnot limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethyleneglycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethyleneglycol). The cationic lipid may be selected from any lipid known in theart such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 andDLin-KC2-DMA.

In one embodiment, the LNP formulations of the polynucleotides, primaryconstructs and/or mmRNA may contain PEG-c-DOMG 3% lipid molar ratio. Inanother embodiment, the LNP formulations polynucleotides, primaryconstructs and/or mmRNA may contain PEG-c-DOMG 1.5% lipid molar ratio.

In one embodiment, the pharmaceutical compositions of thepolynucleotides, primary constructs and/or mmRNA may include at leastone of the PEGylated lipids described in International Publication No.2012099755, herein incorporated by reference.

In one embodiment, the pharmaceutical compositions may be formulated inliposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech,Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutralDOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g.,siRNA delivery for ovarian cancer (Landen et al. Cancer Biology &Therapy 2006 5(12)1708-1713)) and hyaluronan-coated liposomes (QuietTherapeutics, Israel).

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In one embodiment, the internal ester linkage may be located on eitherside of the saturated carbon. Non-limiting examples of reLNPs include,

In one embodiment, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 20120189700 and InternationalPublication No. WO2012099805; herein incorporated by reference in theirentireties). The polymer may encapsulate the nanospecies or partiallyencapsulate the nanospecies. The immunogen may be a recombinant protein,a modified RNA and/or a primary construct described herein. In oneembodiment, the lipid nanoparticle may be formulated for use in avaccine such as, but not limited to, against a pathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limted to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosla tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; herein incorporated by reference in their entirety). Thetransport of nanoparticles may be determined using rates of permeationand/or fluorescent microscopy techniques including, but not limited to,fluorescence recovery after photobleaching (FRAP) and high resolutionmultiple particle tracking (MPT).

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. Non-limiting examples of specificpolymers include poly(caprolactone) (PCL), ethylene vinyl acetatepolymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA),poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA),poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA),poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a co-polymer such as, but not limited to, a block co-polymer, and(poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol))triblock copolymer (see US Publication 20120121718 and US Publication20100003337; herein incorporated by reference in their entireties). Theco-polymer may be a polymer that is generally regarded as safe (GRAS)and the formation of the lipid nanoparticle may be in such a way that nonew chemical entities are created. For example, the lipid nanoparticlemay comprise poloxamers coating PLGA nanoparticles without forming newchemical entities which are still able to rapidly penetrate human mucus(Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; hereinincorporated by reference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, mmRNA, anionic protein(e.g., bovine serum albumin), surfactants (e.g., cationic surfactantssuch as for example dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g.,N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol,sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosinβ4 dornase alfa, neltenexine, erdosteine) and various DNases includingrhDNase. The surface altering agent may be embedded or enmeshed in theparticle's surface or disposed (e.g., by coating, adsorption, covalentlinkage, or other process) on the surface of the lipid nanoparticle.(see US Publication 20100215580 and US Publication 20080166414; hereinincorporated by reference in their entireties).

The mucus penetrating lipid nanoparticles may comprise at least onemmRNA described herein. The mmRNA may be encapsulated in the lipidnanoparticle and/or disposed on the surface of the particle. The mmRNAmay be covalently coupled to the lipid nanoparticle. Formulations ofmucus penetrating lipid nanoparticles may comprise a plurality ofnanoparticles. Further, the formulations may contain particles which mayinteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion which mayincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

In one embodiment, the polynucleotide, primary construct, or mmRNA isformulated as a lipoplex, such as, without limitation, the ATUPLEX™system, the DACC system, the DBTC system and other siRNA-lipoplextechnology from Silence Therapeutics (London, United Kingdom), STEMFECT™from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) orprotamine-based targeted and non-targeted delivery of nucleic acidsacids (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al. IntJ Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 200613:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier etal., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. MicrovascRes 2010 80:286-293Weide et al. J Immunother. 2009 32:498-507; Weide etal. J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther.4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15; Song etal., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl AcadSci USA. 2007 6; 104:4095-4100; deFougerolles Hum Gene Ther. 200819:125-132; all of which are incorporated herein by reference in itsentirety).

In one embodiment such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and MC3-based lipid nanoparticle formulationswhich have been shown to bind to apolipoprotein E and promote bindingand uptake of these formulations into hepatocytes in vivo (Akinc et al.Mol Ther. 2010 18:1357-1364; herein incorporated by reference in itsentirety). Formulations can also be selectively targeted throughexpression of different ligands on their surface as exemplified by, butnot limited by, folate, transferrin, N-acetylgalactosamine (Ga1NAc), andantibody targeted approaches (Kolhatkar et al., Curr Drug DiscovTechnol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 201116:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al.,Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al.,Biomacromolecules. 2011 12:2708-2714Zhao et al., Expert Opin Drug Deliv.2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan etal., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods MolBiol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer etal., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et al., Methods MolBiol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037;Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; all ofwhich are incorporated herein by reference in its entirety).

In one embodiment, the polynucleotide, primary construct, or mmRNA isformulated as a solid lipid nanoparticle. A solid lipid nanoparticle(SLN) may be spherical with an average diameter between 10 to 1000 nm.SLN possess a solid lipid core matrix that can solubilize lipophilicmolecules and may be stabilized with surfactants and/or emulsifiers. Ina further embodiment, the lipid nanoparticle may be a self-assemblylipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp1696-1702; herein incorporated by reference in its entirety).

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotide, primary construct, or mmRNA directed proteinproduction as these formulations may be able to increase celltransfection by the polynucleotide, primary construct, or mmRNA; and/orincrease the translation of encoded protein. One such example involvesthe use of lipid encapsulation to enable the effective systemic deliveryof polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720; hereinincorporated by reference in its entirety). The liposomes, lipoplexes,or lipid nanoparticles may also be used to increase the stability of thepolynucleotide, primary construct, or mmRNA.

Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

The polynucleotide, primary construct, and mmRNA of the invention can beformulated using natural and/or synthetic polymers. Non-limitingexamples of polymers which may be used for delivery include, but are notlimited to, Dynamic POLYCONJUGATE™ formulations from MIRUS® Bio(Madison, Wis.) and Roche Madison (Madison, Wis.), PHASERX™ polymerformulations such as, without limitation, SMARTT POLYMER TECHNOLOGY™(Seattle, Wash.), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical(San Diego, Calif.), chitosan, cyclodextrin from Calando Pharmaceuticals(Pasadena, Calif.), dendrimers and poly(lactic-co-glycolic acid) (PLGA)polymers. RONDEL™ (RNAi/Oligonucleotide Nanoparticle Delivery) polymers(Arrowhead Research Corporation, Pasadena, Calif.) and pH responsiveco-block polymers such as, but not limited to, PHASERX™ (Seattle,Wash.).

A non-limiting example of PLGA formulations include, but are not limitedto, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolvingPLGA in 66% N-methyl-2-pyrrolidone (NMP) and the remainder being aqueoussolvent and leuprolide. Once injected, the PLGA and leuprolide peptideprecipitates into the subcutaneous space).

Many of these polymer approaches have demonstrated efficacy indelivering oligonucleotides in vivo into the cell cytoplasm (reviewed indeFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated byreference in its entirety). Two polymer approaches that have yieldedrobust in vivo delivery of nucleic acids, in this case with smallinterfering RNA (siRNA), are dynamic polyconjugates andcyclodextrin-based nanoparticles. The first of these delivery approachesuses dynamic polyconjugates and has been shown in vivo in mice toeffectively deliver siRNA and silence endogenous target mRNA inhepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887). This particular approach is a multicomponent polymersystem whose key features include a membrane-active polymer to whichnucleic acid, in this case siRNA, is covalently coupled via a disulfidebond and where both PEG (for charge masking) and N-acetylgalactosamine(for hepatocyte targeting) groups are linked via pH-sensitive bonds(Rozema et al., Proc Natl Acad Sci USA. 2007 104:12982-12887). Onbinding to the hepatocyte and entry into the endosome, the polymercomplex disassembles in the low-pH environment, with the polymerexposing its positive charge, leading to endosomal escape andcytoplasmic release of the siRNA from the polymer. Through replacementof the N-acetylgalactosamine group with a mannose group, it was shownone could alter targeting from asialoglycoprotein receptor-expressinghepatocytes to sinusoidal endothelium and Kupffer cells. Another polymerapproach involves using transferrin-targeted cyclodextrin-containingpolycation nanoparticles. These nanoparticles have demonstrated targetedsilencing of the EWS-FLI1 gene product in transferrinreceptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et al.,Cancer Res. 2005 65: 8984-8982) and siRNA formulated in thesenanoparticles was well tolerated in non-human primates (Heidel et al.,Proc Natl Acad Sci USA 2007 104:5715-21). Both of these deliverystrategies incorporate rational approaches using both targeted deliveryand endosomal escape mechanisms.

The polymer formulation can permit the sustained or delayed release ofpolynucleotide, primary construct, or mmRNA (e.g., followingintramuscular or subcutaneous injection). The altered release profilefor the polynucleotide, primary construct, or mmRNA can result in, forexample, translation of an encoded protein over an extended period oftime. The polymer formulation may also be used to increase the stabilityof the polynucleotide, primary construct, or mmRNA. Biodegradablepolymers have been previously used to protect nucleic acids other thanmmRNA from degradation and been shown to result in sustained release ofpayloads in vivo (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887; Sullivan et al., Expert Opin Drug Deliv. 20107:1433-1446; Convertine et al., Biomacromolecules. 2010 Oct. 1; Chu etal., Acc Chem Res. 2012 Jan. 13; Manganiello et al., Biomaterials. 201233:2301-2309; Benoit et al., Biomacromolecules. 2011 12:2708-2714;Singha et al., Nucleic Acid Ther. 2011 2:133-147; deFougerolles Hum GeneTher. 2008 19:125-132; Schaffert and Wagner, Gene Ther. 200816:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 20118:1455-1468; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010464:1067-1070; herein incorporated by reference in its entirety).

In one embodiment, the pharmaceutical compositions may be sustainedrelease formulations. In a further embodiment, the sustained releaseformulations may be for subcutaneous delivery. Sustained releaseformulations may include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego Calif.),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,Ga.). TISSELL® (Baxter International, Inc Deerfield, Ill.), PEG-basedsealants, and COSEAL® (Baxter International, Inc Deerfield, Ill.),

As a non-limiting example modified mRNA may be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradeable,biocompatible polymers which are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine deivce; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C. PEG-based surgical sealants comprise two syntheticPEG components mixed in a delivery device which can be prepared in oneminute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE®and natural polymers are capable of in-situ gelation at the site ofadministration. They have been shown to interact with protein andpeptide therapeutic candidates through ionic ineraction to provide astabilizing effect.

Polymer formulations can also be selectively targeted through expressionof different ligands as exemplified by, but not limited by, folate,transferrin, and N-acetylgalactosamine (Ga1NAc) (Benoit et al.,Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad SciUSA. 2007 104:12982-12887; Davis, Mol Pharm. 2009 6:659-668; Davis,Nature 2010 464:1067-1070; herein incorporated by reference in itsentirety).

The modified nucleic acid, and mmRNA of the invention may be formulatedwith or in a polymeric compound. The polymer may include at least onepolymer such as, but not limited to, polyethylene glycol (PEG),poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer,biodegradable cationic lipopolymer, polyethyleneimine (PEI),cross-linked branched poly(alkylene imines), a polyamine derivative, amodified poloxamer, a biodegradable polymer, biodegradable blockcopolymer, biodegradable random copolymer, biodegradable polyestercopolymer, biodegradable polyester block copolymer, biodegradablepolyester block random copolymer, linear biodegradable copolymer,poly[α-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradablecross-linked cationic multi-block copolymers or combinations thereof.

As a non-limiting example, the modified nucleic acid or mmRNA of theinvention may be formulated with the polymeric compound of PEG graftedwith PLL as described in U.S. Pat. No. 6,177,274 herein incorporated byreference in its entirety. The formulation may be used for transfectingcells in vitro or for in vivo delivery of the modified nucleic acid andmmRNA. In another example, the modified nucleic acid and mmRNA may besuspended in a solution or medium with a cationic polymer, in a drypharmaceutical composition or in a solution that is capable of beingdried as described in U.S. Pub. Nos. 20090042829 and 20090042825 each ofwhich are herein incorporated by reference in their entireties.

A polyamine derivative may be used to deliver nucleic acids or to treatand/or prevent a disease or to be included in an implantable orinjectable device (U.S. Pub. No. 20100260817 herein incorporated byreference in its entirety). As a non-limiting example, a pharmaceuticalcomposition may include the modified nucleic acids and mmRNA and thepolyamine derivative described in U.S. Pub. No. 20100260817 (thecontents of which are incorporated herein by reference in its entirety.

For example, the modified nucleic acid or mmRNA of the invention may beformulated in a pharmaceutical compound including a poly(alkyleneimine), a biodegradable cationic lipopolymer, a biodegradable blockcopolymer, a biodegradable polymer, or a biodegradable random copolymer,a biodegradable polyester block copolymer, a biodegradable polyesterpolymer, a biodegradable polyester random copolymer, a linearbiodegradable copolymer, PAGA, a biodegradable cross-linked cationicmulti-block copolymer or combinations thereof. The biodegradablecationic lipopolymer may be made my methods known in the art and/ordescribed in U.S. Pat. No. 6,696,038, U.S. App. Nos. 20030073619 and20040142474 which is herein incorporated by reference in theirentireties. The poly(alkylene imine) may be made using methods known inthe art and/or as described in U.S. Pub. No. 20100004315, hereinincorporated by reference in its entirety. The biodegradabale polymer,biodegradable block copolymer, the biodegradable random copolymer,biodegradable polyester block copolymer, biodegradable polyesterpolymer, or biodegradable polyester random copolymer may be made usingmethods known in the art and/or as described in U.S. Pat. Nos. 6,517,869and 6,267,987, the contents of which are each incorporated herein byreference in its entirety. The linear biodegradable copolymer may bemade using methods known in the art and/or as described in U.S. Pat. No.6,652,886. The PAGA polymer may be made using methods known in the artand/or as described in U.S. Pat. No. 6,217,912 herein incorporated byreference in its entirety. The PAGA polymer may be copolymerized to forma copolymer or block copolymer with polymers such as but not limited to,poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines,polylactides and poly(lactide-co-glycolides). The biodegradablecross-linked cationic multi-block copolymers may be made my methodsknown in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S.Pub. No. 2012009145 herein incorporated by reference in theirentireties. For example, the multi-block copolymers may be synthesizedusing linear polyethyleneimine (LPEI) blocks which have distinctpatterns as compared to branched polyethyleneimines. Further, thecomposition or pharmaceutical composition may be made by the methodsknown in the art, described herein, or as described in U.S. Pub. No.20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912 hereinincorporated by reference in their entireties.

As described in U.S. Pub. No. 20100004313, herein incorporated byreference in its entirety, a gene delivery composition may include anucleotide sequence and a poloxamer. For example, the modified nucleicacid and mmRNA of the present inveition may be used in a gene deliverycomposition with the poloxamer described in U.S. Pub. No. 20100004313.

In one embodiment, the polymer formulation of the present invention maybe stabilized by contacting the polymer formulation, which may include acationic carrier, with a cationic lipopolymer which may be covalentlylinked to cholesterol and polyethylene glycol groups. The polymerformulation may be contacted with a cationic lipopolymer using themethods described in U.S. Pub. No. 20090042829 herein incorporated byreference in its entirety. The cationic carrier may include, but is notlimited to, polyethylenimine, poly(trimethylenimine),poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine,dideoxy-diamino-b-cyclodextrin, spermine, spermidine,poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine),poly(arginine), cationized gelatin, dendrimers, chitosan,1,2-Dioleoyl-3-Trimethylammonium-Propane(DOTAP),N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride(DOTIM),2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA),3B—[N—(N′,N′-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride(DC-Cholesterol HCl) diheptadecylamidoglycyl spermidine (DOGS),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) andcombinations thereof.

The polynucleotide, primary construct, and mmRNA of the invention canalso be formulated as a nanoparticle using a combination of polymers,lipids, and/or other biodegradable agents, such as, but not limited to,calcium phosphate. Components may be combined in a core-shell, hybrid,and/or layer-by-layer architecture, to allow for fine-tuning of thenanoparticle so to delivery of the polynucleotide, primary construct andmmRNA may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller etal., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug DelivRev. 2011 63:748-761; Endres et al., Biomaterials. 2011 32:7721-7731; Suet al., Mol Pharm. 2011 Jun. 6; 8(3):774-87; herein incorporated byreference in its entirety).

Biodegradable calcium phosphate nanoparticles in combination with lipidsand/or polymers have been shown to deliver polynucleotides, primaryconstructs and mmRNA in vivo. In one embodiment, a lipid coated calciumphosphate nanoparticle, which may also contain a targeting ligand suchas anisamide, may be used to deliver the polynucleotide, primaryconstruct and mmRNA of the present invention. For example, toeffectively deliver siRNA in a mouse metastatic lung model a lipidcoated calcium phosphate nanoparticle was used (Li et al., J Contr Rel.2010 142: 416-421; Li et al., J Contr Rel. 2012 158:108-114; Yang etal., Mol Ther. 2012 20:609-615). This delivery system combines both atargeted nanoparticle and a component to enhance the endosomal escape,calcium phosphate, in order to improve delivery of the siRNA.

In one embodiment, calcium phosphate with a PEG-polyanion blockcopolymer may be used to delivery polynucleotides, primary constructsand mmRNA (Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa etal., J Contr Rel. 2006 111:368-370).

In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle todeliver the polynucleotides, primary constructs and mmRNA of the presentinvention. The PEG-charge-conversional polymer may improve upon thePEG-polyanion block copolymers by being cleaved into a polycation atacidic pH, thus enhancing endosomal escape.

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001). The complexation, delivery, and internalization of thepolymeric nanoparticles can be precisely controlled by altering thechemical composition in both the core and shell components of thenanoparticle. For example, the core-shell nanoparticles may efficientlydeliver siRNA to mouse hepatocytes after they covalently attachcholesterol to the nanoparticle.

In one embodiment, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer containg PEG may be used to delivery ofthe polynucleotide, primary construct and mmRNA of the presentinvention. As a non-limiting example, in mice bearing aluciferease-expressing tumor, it was determined that thelipid-polymer-lipid hybrid nanoparticle significantly suppressedluciferase expression, as compared to a conventional lipoplex (Shi etal, Angew Chem Int Ed. 2011 50:7027-7031).

Peptides and Proteins

The polynucleotide, primary construct, and mmRNA of the invention can beformulated with peptides and/or proteins in order to increasetransfection of cells by the polynucleotide, primary construct, ormmRNA. In one embodiment, peptides such as, but not limited to, cellpenetrating peptides and proteins and peptides that enable intracellulardelivery may be used to deliver pharmaceutical formulations. Anon-limiting example of a cell penetrating peptide which may be usedwith the pharmaceutical formulations of the present invention includes acell-penetrating peptide sequence attached to polycations thatfacilitates delivery to the intracellular space, e.g., HIV-derived TATpeptide, penetratins, transportans, or hCT derived cell-penetratingpeptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel,Cell-Penetrating Peptides: Processes and Applications (CRC Press, BocaRaton Fla., 2002); El-Andaloussi et al., Curr. Pharm. Des.11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life Sci.62(16):1839-49 (2005), all of which are incorporated herein byreference). The compositions can also be formulated to include a cellpenetrating agent, e.g., liposomes, which enhance delivery of thecompositions to the intracellular space. Polynucleotides, primaryconstructs, and mmRNA of the invention may be complexed to peptidesand/or proteins such as, but not limited to, peptides and/or proteinsfrom Aileron Therapeutics (Cambridge, Mass.) and Permeon Biologics(Cambridge, Mass.) in order to enable intracellular delivery (Cronicanet al., ACS Chem. Biol. 2010 5:747-752; McNaughton et al., Proc. Natl.Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des. 200973:3-6; Verdine and Hilinski, Methods Enzymol. 2012; 503:3-33; all ofwhich are herein incorporated by reference in its entirety).

In one embodiment, the cell-penetrating polypeptide may comprise a firstdomain and a second domain. The first domain may comprise a superchargedpolypeptide. The second domain may comprise a protein-binding partner.As used herein, “protein-binding partner” includes, but are not limitedto, antibodies and functional fragments thereof, scaffold proteins, orpeptides. The cell-penetrating polypeptide may further comprise anintracellular binding partner for the protein-binding partner. Thecell-penetrating polypeptide may be capable of being secreted from acell where the polynucleotide, primary construct, or mmRNA may beintroduced.

Formulations of the including peptides or proteins may be used toincrease cell transfection by the polynucleotide, primary construct, ormmRNA, alter the biodistribution of the polynucleotide, primaryconstruct, or mmRNA (e.g., by targeting specific tissues or cell types),and/or increase the translation of encoded protein.

Cells

The polynucleotide, primary construct, and mmRNA of the invention can betransfected ex vivo into cells, which are subsequently transplanted intoa subject. As non-limiting examples, the pharmaceutical compositions mayinclude red blood cells to deliver modified RNA to liver and myeloidcells, virosomes to deliver modified RNA in virus-like particles (VLPs),and electroporated cells such as, but not limited to, from MAXCYTE®(Gaithersburg, Md.) and from ERYTECH® (Lyon, France) to deliver modifiedRNA. Examples of use of red blood cells, viral particles andelectroporated cells to deliver payloads other than mmRNA have beendocumented (Godfrin et al., Expert Opin Biol Ther. 2012 12:127-133; Fanget al., Expert Opin Biol Ther. 2012 12:385-389; Hu et al., Proc NatlAcad Sci USA. 2011 108:10980-10985; Lund et al., Pharm Res. 201027:400-420; Huckriede et al., J Liposome Res. 2007; 17:39-47; Cusi, HumVaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all ofwhich are herein incorporated by reference in its entirety).

Cell-based formulations of the polynucleotide, primary construct, andmmRNA of the invention may be used to ensure cell transfection (e.g., inthe cellular carrier), alter the biodistribution of the polynucleotide,primary construct, or mmRNA (e.g., by targeting the cell carrier tospecific tissues or cell types), and/or increase the translation ofencoded protein.

A variety of methods are known in the art and suitable for introductionof nucleic acid into a cell, including viral and non-viral mediatedtechniques. Examples of typical non-viral mediated techniques include,but are not limited to, electroporation, calcium phosphate mediatedtransfer, nucleofection, sonoporation, heat shock, magnetofection,liposome mediated transfer, microinjection, microproj ectile mediatedtransfer (nanoparticles), cationic polymer mediated transfer(DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like)or cell fusion.

The technique of sonoporation, or cellular sonication, is the use ofsound (e.g., ultrasonic frequencies) for modifying the permeability ofthe cell plasma membrane. Sonoporation methods are known to those in theart and are used to deliver nucleic acids in vivo (Yoon and Park, ExpertOpin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr PharmBiotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 200714:465-475; all herein incorporated by reference in their entirety).Sonoporation methods are known in the art and are also taught forexample as it relates to bacteria in US Patent Publication 20100196983and as it relates to other cell types in, for example, US PatentPublication 20100009424, each of which are incorporated herein byreference in their entirety.

Electroporation techniques are also well known in the art and are usedto deliver nucleic acids in vivo and clinically (Andre et al., Curr GeneTher. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 201010:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all hereinincorporated by reference in their entirety). In one embodiment,polynucleotides, primary constructs or mmRNA may be delivered byelectroporation as described in Example 8.

Hyaluronidase

The intramuscular or subcutaneous localized injection of polynucleotide,primary construct, or mmRNA of the invention can include hyaluronidase,which catalyzes the hydrolysis of hyaluronan. By catalyzing thehydrolysis of hyaluronan, a constituent of the interstitial barrier,hyaluronidase lowers the viscosity of hyaluronan, thereby increasingtissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440;herein incorporated by reference in its entirety). It is useful to speedtheir dispersion and systemic distribution of encoded proteins producedby transfected cells. Alternatively, the hyaluronidase can be used toincrease the number of cells exposed to a polynucleotide, primaryconstruct, or mmRNA of the invention administered intramuscularly orsubcutaneously.

Nanoparticle Mimics

The polynucleotide, primary construct or mmRNA of the invention may beencapsulated within and/or absorbed to a nanoparticle mimic. Ananoparticle mimic can mimic the delivery function organisms orparticles such as, but not limited to, pathogens, viruses, bacteria,fungus, parasites, prions and cells. As a non-limiting example thepolynucleotide, primary construct or mmRNA of the invention may beencapsulated in a non-viron particle which can mimic the deliveryfunction of a virus (see International Pub. No. WO2012006376 hereinincorporated by reference in its entirety).

Nanotubes

The polynucleotides, primary constructs or mmRNA of the invention can beattached or otherwise bound to at least one nanotube such as, but notlimited to, rosette nanotubes, rosette nanotubes having twin bases witha linker, carbon nanotubes and/or single-walled carbon nanotubes, Thepolynucleotides, primary constructs or mmRNA may be bound to thenanotubes through forces such as, but not limited to, steric, ionic,covalent and/or other forces.

In one embodiment, the nanotube can release one or more polynucleotides,primary constructs or mmRNA into cells. The size and/or the surfacestructure of at least one nanotube may be altered so as to govern theinteraction of the nanotubes within the body and/or to attach or bind tothe polynucleotides, primary constructs or mmRNA disclosed herein. Inone embodiment, the building block and/or the functional groups attachedto the building block of the at least one nanotube may be altered toadjust the dimensions and/or properties of the nanotube. As anon-limiting example, the length of the nanotubes may be altered tohinder the nanotubes from passing through the holes in the walls ofnormal blood vessels but still small enough to pass through the largerholes in the blood vessels of tumor tissue.

In one embodiment, at least one nanotube may also be coated withdelivery enhancing compounds including polymers, such as, but notlimited to, polyethylene glycol. In another embodiment, at least onenanotube and/or the polynucleotides, primary constructs or mmRNA may bemixed with pharmaceutically acceptable excipients and/or deliveryvehicles.

In one embodiment, the polynucleotides, primary constructs or mmRNA areattached and/or otherwise bound to at least one rosette nanotube. Therosette nanotubes may be formed by a process known in the art and/or bythe process described in International Publication No. WO2012094304,herein incorporated by reference in its entirety. At least onepolynucleotide, primary construct and/or mmRNA may be attached and/orotherwise bound to at least one rosette nanotube by a process asdescribed in International Publication No. WO2012094304, hereinincorporated by reference in its entirety, where rosette nanotubes ormodules forming rosette nanotubes are mixed in aqueous media with atleast one polynucleotide, primary construct and/or mmRNA underconditions which may cause at least one polynucleotide, primaryconstruct or mmRNA to attach or otherwise bind to the rosette nanotubes.

Conjugates

The polynucleotides, primary constructs, and mmRNA of the inventioninclude conjugates, such as a polynucleotide, primary construct, ormmRNA covalently linked to a carrier or targeting group, or includingtwo encoding regions that together produce a fusion protein (e.g.,bearing a targeting group and therapeutic protein or peptide).

The conjugates of the invention include a naturally occurring substance,such as a protein (e.g., human serum albumin (HSA), low-densitylipoprotein (LDL), high-density lipoprotein (HDL), or globulin); ancarbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin,cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be arecombinant or synthetic molecule, such as a synthetic polymer, e.g., asynthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examplesof polyamino acids include polyamino acid is a polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Representative U.S. patents that teach the preparation of polynucleotideconjugates, particularly to RNA, include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and U.S. Pat. Nos. 5,688,941;6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; eachof which is herein incorporated by reference in their entireties.

In one embodiment, the conjugate of the present invention may functionas a carrier for the modified nucleic acids and mmRNA of the presentinvention. The conjugate may comprise a cationic polymer such as, butnot limited to, polyamine, polylysine, polyalkylenimine, andpolyethylenimine which may be grafted to with poly(ethylene glycol). Asa non-limiting example, the conjugate may be similar to the polymericconjugate and the method of synthesizing the polymeric conjugatedescribed in U.S. Pat. No. 6,586,524 herein incorporated by reference inits entirety.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas a cancer cell, endothelial cell, or bone cell. Targeting groups mayalso include hormones and hormone receptors. They can also includenon-peptidic species, such as lipids, lectins, carbohydrates, vitamins,cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,multivalent fucose, or aptamers. The ligand can be, for example, alipopolysaccharide, or an activator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, Ga1NAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein.

In one embodiment, pharmaceutical compositions of the present inventionmay include chemical modifications such as, but not limited to,modifications similar to locked nucleic acids.

Representative U.S. Patents that teach the preparation of locked nucleicacid (LNA) such as those from Santaris, include, but are not limited to,the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is hereinincorporated by reference in its entirety.

Representative U.S. patents that teach the preparation of PNA compoundsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found, for example, in Nielsenet al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include polynucleotides,primary constructs or mmRNA with phosphorothioate backbones andoligonucleosides with other modified backbones, and in particular—CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) orMMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂—and—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone isrepresented as —O—P(O)₂—O—CH₂—] of the above-referenced U.S. Pat. No.5,489,677, and the amide backbones of the above-referenced U.S. Pat. No.5,602,240. In some embodiments, the polynucleotides featured herein havemorpholino backbone structures of the above-referenced U.S. Pat. No.5,034,506.

Modifications at the 2′ position may also aid in delivery. Preferably,modifications at the 2′ position are not located in a polypeptide-codingsequence, i.e., not in a translatable region. Modifications at the 2′position may be located in a 5′UTR, a 3′UTR and/or a tailing region.Modifications at the 2′ position can include one of the following at the2′ position: H (i.e., 2′-deoxy); F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, the polynucleotides,primary constructs or mmRNA include one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties, or a group for improving the pharmacodynamicproperties, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below. Othermodifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions, particularly the 3′ position of the sugar onthe 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ positionof 5′ terminal nucleotide. Polynucleotides of the invention may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920 and each of which isherein incorporated by reference.

In still other embodiments, the polynucleotide, primary construct, ormmRNA is covalently conjugated to a cell penetrating polypeptide. Thecell-penetrating peptide may also include a signal sequence. Theconjugates of the invention can be designed to have increased stability;increased cell transfection; and/or altered the biodistribution (e.g.,targeted to specific tissues or cell types).

Self-Assembled Nucleic Acid Nanoparticles

Self-assembled nanoparticles have a well-defined size which may beprecisely controlled as the nucleic acid strands may be easilyreprogrammable. For example, the optimal particle size for acancer-targeting nanodelivery carrier is 20-100 nm as a diameter greaterthan 20 nm avoids renal clearance and enhances delivery to certaintumors through enhanced permeability and retention effect. Usingself-assembled nucleic acid nanoparticles a single uniform population insize and shape having a precisely controlled spatial orientation anddensity of cancer-targeting ligands for enhanced delivery. As anon-limiting example, oligonucleotide nanoparticles were prepared usingprogrammable self-assembly of short DNA fragments and therapeuticsiRNAs. These nanoparticles are molecularly identical with controllableparticle size and target ligand location and density. The DNA fragmentsand siRNAs self-assembled into a one-step reaction to generate DNA/siRNAtetrahedral nanoparticles for targeted in vivo delivery. (Lee et al.,Nature Nanotechnology 2012 7:389-393).

Excipients

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference) discloses various excipientsused in formulating pharmaceutical compositions and known techniques forthe preparation thereof. Except insofar as any conventional excipientmedium is incompatible with a substance or its derivatives, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical compositions.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (VEEGUM®), sodium lauryl sulfate, quaternary ammoniumcompounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEENn®60],polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate[SPAN®40], sorbitan monostearate [Span®60], sorbitan tristearate[Span®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER® 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural andsynthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GLYDANTPLUS®, PHENONIP, methylparaben, GERMALL 115, GERMABEN®II, NEOLONE™,KATHON™, and/or EUXYL®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Delivery

The present disclosure encompasses the delivery of polynucleotides,primary constructs or mmRNA for any of therapeutic, pharmaceutical,diagnostic or imaging by any appropriate route taking into considerationlikely advances in the sciences of drug delivery. Delivery may be nakedor formulated.

Naked Delivery

The polynucleotides, primary constructs or mmRNA of the presentinvention may be delivered to a cell naked. As used herein in, “naked”refers to delivering polynucleotides, primary constructs or mmRNA freefrom agents which promote transfection. For example, thepolynucleotides, primary constructs or mmRNA delivered to the cell maycontain no modifications. The naked polynucleotides, primary constructsor mmRNA may be delivered to the cell using routes of administrationknown in the art and described herein.

Formulated Delivery

The polynucleotides, primary constructs or mmRNA of the presentinvention may be formulated, using the methods described herein. Theformulations may contain polynucleotides, primary constructs or mmRNAwhich may be modified and/or unmodified. The formulations may furtherinclude, but are not limited to, cell penetration agents, apharmaceutically acceptable carrier, a delivery agent, a bioerodible orbiocompatible polymer, a solvent, and a sustained-release deliverydepot. The formulated polynucleotides, primary constructs or mmRNA maybe delivered to the cell using routes of administration known in the artand described herein.

The compositions may also be formulated for direct delivery to an organor tissue in any of several ways in the art including, but not limitedto, direct soaking or bathing, via a catheter, by gels, powder,ointments, creams, gels, lotions, and/or drops, by using substrates suchas fabric or biodegradable materials coated or impregnated with thecompositions, and the like.

Administration

The polynucleotides, primary constructs or mmRNA of the presentinvention may be administered by any route which results in atherapeutically effective outcome. These include, but are not limited toenteral, gastroenteral, epidural, oral, transdermal, epidural(peridural), intracerebral (into the cerebrum), intracerebroventricular(into the cerebral ventricles), epicutaneous (application onto theskin), intradermal, (into the skin itself), subcutaneous (under theskin), nasal administration (through the nose), intravenous (into avein), intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection, (into the base of thepenis), intravaginal administration, intrauterine, extra-amnioticadministration, transdermal (diffusion through the intact skin forsystemic distribution), transmucosal (diffusion through a mucousmembrane), insufflation (snorting), sublingual, sublabial, enema, eyedrops (onto the conjunctiva), or in ear drops. In specific embodiments,compositions may be administered in a way which allows them cross theblood-brain barrier, vascular barrier, or other epithelial barrier.Non-limiting routes of administration for the polynucleotides, primaryconstructs or mmRNA of the present invention are described below.

Parenteral and Injectible Administration

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as CREMOPHOR®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Oral Administration

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g. starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.glycerol), disintegrating agents (e.g. agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g. paraffin), absorptionaccelerators (e.g. quaternary ammonium compounds), wetting agents (e.g.cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin andbentonite clay), and lubricants (e.g. talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate), andmixtures thereof. In the case of capsules, tablets and pills, the dosageform may comprise buffering agents.

Topical or Transdermal Administration

As described herein, compositions containing the polynucleotides,primary constructs or mmRNA of the invention may be formulated foradministration topically. The skin may be an ideal target site fordelivery as it is readily accessible. Gene expression may be restrictednot only to the skin, potentially avoiding nonspecific toxicity, butalso to specific layers and cell types within the skin.

The site of cutaneous expression of the delivered compositions willdepend on the route of nucleic acid delivery. Three routes are commonlyconsidered to deliver polynucleotides, primary constructs or mmRNA tothe skin: (i) topical application (e.g. for local/regional treatmentand/or cosmetic applications); (ii) intradermal injection (e.g. forlocal/regional treatment and/or cosmetic applications); and (iii)systemic delivery (e.g. for treatment of dermatologic diseases thataffect both cutaneous and extracutaneous regions). Polynucleotides,primary constructs or mmRNA can be delivered to the skin by severaldifferent approaches known in the art. Most topical delivery approacheshave been shown to work for delivery of DNA, such as but not limited to,topical application of non-cationic liposome-DNA complex, cationicliposome-DNA complex, particle-mediated (gene gun), puncture-mediatedgene transfections, and viral delivery approaches. After delivery of thenucleic acid, gene products have been detected in a number of differentskin cell types, including, but not limited to, basal keratinocytes,sebaceous gland cells, dermal fibroblasts and dermal macrophages.

In one embodiment, the invention provides for a variety of dressings(e.g., wound dressings) or bandages (e.g., adhesive bandages) forconveniently and/or effectively carrying out methods of the presentinvention. Typically dressing or bandages may comprise sufficientamounts of pharmaceutical compositions and/or polynucleotides, primaryconstructs or mmRNA described herein to allow a user to perform multipletreatments of a subject(s).

In one embodiment, the invention provides for the polynucleotides,primary constructs or mmRNA compositions to be delivered in more thanone injection.

In one embodiment, before topical and/or transdermal administration atleast one area of tissue, such as skin, may be subjected to a deviceand/or solution which may increase permeability. In one embodiment, thetissue may be subjected to an abrasion device to increase thepermeability of the skin (see U.S. Patent Publication No. 20080275468,herein incorporated by reference in its entirety). In anotherembodiment, the tissue may be subjected to an ultrasound enhancementdevice. An ultrasound enhancement device may include, but is not limitedto, the devices described in U.S. Publication No. 20040236268 and U.S.Pat. Nos. 6,491,657 and 6,234,990; herein incorporated by reference intheir entireties. Methods of enhancing the permeability of tissue aredescribed in U.S. Publication Nos. 20040171980 and 20040236268 and U.S.Pat. No. 6,190,315; herein incorporated by reference in theirentireties.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein.The permeability of skin may be measured by methods known in the artand/or described in U.S. Pat. No. 6,190,315, herein incorporated byreference in its entirety. As a non-limiting example, a modified mRNAformulation may be delivered by the drug delivery methods described inU.S. Pat. No. 6,190,315, herein incorporated by reference in itsentirety.

In another non-limiting example tissue may be treated with a eutecticmixture of local anesthetics (EMLA) cream before, during and/or afterthe tissue may be subjected to a device which may increase permeability.Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated byreference in its entirety) showed that using the EMLA cream incombination with a low energy, an onset of superficial cutaneousanalgesia was seen as fast as 5 minutes after a pretreatment with a lowenergy ultrasound.

In one embodiment, enhancers may be applied to the tissue before,during, and/or after the tissue has been treated to increasepermeability. Enhancers include, but are not limited to, transportenhancers, physical enhancers, and cavitation enhancers. Non-limitingexamples of enhancers are described in U.S. Pat. No. 6,190,315, hereinincorporated by reference in its entirety.

In one embodiment, a device may be used to increase permeability oftissue before delivering formulations of modified mRNA described herein,which may further contain a substance that invokes an immune response.In another non-limiting example, a formulation containing a substance toinvoke an immune response may be delivered by the methods described inU.S. Publication Nos. 20040171980 and 20040236268; herein incorporatedby reference in their entireties.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present inventioncontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.

Topically-administrable formulations may, for example, comprise fromabout 0.1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

Depot Administration

As described herein, in some embodiments, the composition is formulatedin depots for extended release. Generally, a specific organ or tissue (a“target tissue”) is targeted for administration.

In some aspects of the invention, the polynucleotides, primaryconstructs or mmRNA are spatially retained within or proximal to atarget tissue. Provided are method of providing a composition to atarget tissue of a mammalian subject by contacting the target tissue(which contains one or more target cells) with the composition underconditions such that the composition, in particular the nucleic acidcomponent(s) of the composition, is substantially retained in the targettissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90,95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of thecomposition is retained in the target tissue. Advantageously, retentionis determined by measuring the amount of the nucleic acid present in thecomposition that enters one or more target cells. For example, at least1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9,99.99 or greater than 99.99% of the nucleic acids administered to thesubject are present intracellularly at a period of time followingadministration. For example, intramuscular injection to a mammaliansubject is performed using an aqueous composition containing aribonucleic acid and a transfection reagent, and retention of thecomposition is determined by measuring the amount of the ribonucleicacid present in the muscle cells.

Aspects of the invention are directed to methods of providing acomposition to a target tissue of a mammalian subject, by contacting thetarget tissue (containing one or more target cells) with the compositionunder conditions such that the composition is substantially retained inthe target tissue. The composition contains an effective amount of apolynucleotides, primary constructs or mmRNA such that the polypeptideof interest is produced in at least one target cell. The compositionsgenerally contain a cell penetration agent, although “naked” nucleicacid (such as nucleic acids without a cell penetration agent or otheragent) is also contemplated, and a pharmaceutically acceptable carrier.

In some circumstances, the amount of a protein produced by cells in atissue is desirably increased. Preferably, this increase in proteinproduction is spatially restricted to cells within the target tissue.Thus, provided are methods of increasing production of a protein ofinterest in a tissue of a mammalian subject. A composition is providedthat contains polynucleotides, primary constructs or mmRNA characterizedin that a unit quantity of composition has been determined to producethe polypeptide of interest in a substantial percentage of cellscontained within a predetermined volume of the target tissue.

In some embodiments, the composition includes a plurality of differentpolynucleotides, primary constructs or mmRNA, where one or more than oneof the polynucleotides, primary constructs or mmRNA encodes apolypeptide of interest. Optionally, the composition also contains acell penetration agent to assist in the intracellular delivery of thecomposition. A determination is made of the dose of the compositionrequired to produce the polypeptide of interest in a substantialpercentage of cells contained within the predetermined volume of thetarget tissue (generally, without inducing significant production of thepolypeptide of interest in tissue adjacent to the predetermined volume,or distally to the target tissue). Subsequent to this determination, thedetermined dose is introduced directly into the tissue of the mammaliansubject.

In one embodiment, the invention provides for the polynucleotides,primary constructs or mmRNA to be delivered in more than one injectionor by split dose injections.

In one embodiment, the invention may be retained near target tissueusing a small disposable drug reservoir or patch pump. Non-limitingexamples of patch pumps include those manufactured and/or sold by BD®(Franklin Lakes, N.J.), Insulet Corporation (Bedford, Mass.), SteadyMedTherapeutics (San Francisco, Calif.), Medtronic (Minneapolis, Minn.),UniLife (York, Pa.), Valeritas (Bridgewater, N.J.), and SpringLeafTherapeutics (Boston, Mass.).

Pulmonary Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions aresuitably in the form of dry powders for administration using a devicecomprising a dry powder reservoir to which a stream of propellant may bedirected to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further comprise additional ingredients such as a liquidnon-ionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles comprising theactive ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Intranasal, nasal and buccal Administration

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 um to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e. by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

Ophthalmic Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other ophthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis invention.

Payload Administration: Detectable Agents and Therapeutic Agents

The polynucleotides, primary constructs or mmRNA described herein can beused in a number of different scenarios in which delivery of a substance(the “payload”) to a biological target is desired, for example deliveryof detectable substances for detection of the target, or delivery of atherapeutic agent. Detection methods can include, but are not limitedto, both imaging in vitro and in vivo imaging methods, e.g.,immunohistochemistry, bioluminescence imaging (BLI), Magnetic ResonanceImaging (MRI), positron emission tomography (PET), electron microscopy,X-ray computed tomography, Raman imaging, optical coherence tomography,absorption imaging, thermal imaging, fluorescence reflectance imaging,fluorescence microscopy, fluorescence molecular tomographic imaging,nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging,photoacoustic imaging, lab assays, or in any situation wheretagging/staining/imaging is required.

The polynucleotides, primary constructs or mmRNA can be designed toinclude both a linker and a payload in any useful orientation. Forexample, a linker having two ends is used to attach one end to thepayload and the other end to the nucleobase, such as at the C-7 or C-8positions of the deaza-adenosine or deaza-guanosine or to the N-3 or C-5positions of cytosine or uracil. The polynucleotide of the invention caninclude more than one payload (e.g., a label and a transcriptioninhibitor), as well as a cleavable linker. In one embodiment, themodified nucleotide is a modified 7-deaza-adenosine triphosphate, whereone end of a cleavable linker is attached to the C7 position of7-deaza-adenine, the other end of the linker is attached to an inhibitor(e.g., to the C5 position of the nucleobase on a cytidine), and a label(e.g., Cy5) is attached to the center of the linker (see, e.g., compound1 of A*pCp C5 Parg Capless in FIG. 5 and columns 9 and 10 of U.S. Pat.No. 7,994,304, incorporated herein by reference). Upon incorporation ofthe modified 7-deaza-adenosine triphosphate to an encoding region, theresulting polynucleotide having a cleavable linker attached to a labeland an inhibitor (e.g., a polymerase inhibitor). Upon cleavage of thelinker (e.g., with reductive conditions to reduce a linker having acleavable disulfide moiety), the label and inhibitor are released.Additional linkers and payloads (e.g., therapeutic agents, detectablelabels, and cell penetrating payloads) are described herein.

Scheme 12 below depicts an exemplary modified nucleotide wherein thenucleobase, adenine, is attached to a linker at the C-7 carbon of7-deaza adenine. In addition, Scheme 12 depicts the modified nucleotidewith the linker and payload, e.g., a detectable agent, incorporated ontothe 3′ end of the mRNA. Disulfide cleavage and 1,2-addition of the thiolgroup onto the propargyl ester releases the detectable agent. Theremaining structure (depicted, for example, as pApC5Parg in Scheme 12)is the inhibitor. The rationale for the structure of the modifiednucleotides is that the tethered inhibitor sterically interferes withthe ability of the polymerase to incorporate a second base. Thus, it iscritical that the tether be long enough to affect this function and thatthe inhibiter be in a stereochemical orientation that inhibits orprohibits second and follow on nucleotides into the growingpolynucleotide strand.

For example, the polynucleotides, primary constructs or mmRNA describedherein can be used in reprogramming induced pluripotent stem cells (iPScells), which can directly track cells that are transfected compared tototal cells in the cluster. In another example, a drug that may beattached to the polynucleotides, primary constructs or mmRNA via alinker and may be fluorescently labeled can be used to track the drug invivo, e.g. intracellularly. Other examples include, but are not limitedto, the use of a polynucleotides, primary constructs or mmRNA inreversible drug delivery into cells.

The polynucleotides, primary constructs or mmRNA described herein can beused in intracellular targeting of a payload, e.g., detectable ortherapeutic agent, to specific organelle. Exemplary intracellulartargets can include, but are not limited to, the nuclear localizationfor advanced mRNA processing, or a nuclear localization sequence (NLS)linked to the mRNA containing an inhibitor.

In addition, the polynucleotides, primary constructs or mmRNA describedherein can be used to deliver therapeutic agents to cells or tissues,e.g., in living animals. For example, the polynucleotides, primaryconstructs or mmRNA described herein can be used to deliver highly polarchemotherapeutics agents to kill cancer cells. The polynucleotides,primary constructs or mmRNA attached to the therapeutic agent through alinker can facilitate member permeation allowing the therapeutic agentto travel into a cell to reach an intracellular target.

In another example, the polynucleotides, primary constructs or mmRNA canbe attached to the polynucleotides, primary constructs or mmRNA a viralinhibitory peptide (VIP) through a cleavable linker. The cleavablelinker can release the VIP and dye into the cell. In another example,the polynucleotides, primary constructs or mmRNA can be attached throughthe linker to an ADP-ribosylate, which is responsible for the actions ofsome bacterial toxins, such as cholera toxin, diphtheria toxin, andpertussis toxin. These toxin proteins are ADP-ribosyltransferases thatmodify target proteins in human cells. For example, cholera toxinADP-ribosylates G proteins modifies human cells by causing massive fluidsecretion from the lining of the small intestine, which results inlife-threatening diarrhea.

In some embodiments, the payload may be a therapeutic agent such as acytotoxin, radioactive ion, chemotherapeutic, or other therapeuticagent. A cytotoxin or cytotoxic agent includes any agent that may bedetrimental to cells. Examples include, but are not limited to, taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, teniposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids,e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein inits entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092,5,585,499, and 5,846,545, all of which are incorporated herein byreference), and analogs or homologs thereof. Radioactive ions include,but are not limited to iodine (e.g., iodine 125 or iodine 131),strontium 89, phosphorous, palladium, cesium, iridium, phosphate,cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine(BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine, vinblastine, taxol and maytansinoids).

In some embodiments, the payload may be a detectable agent, such asvarious organic small molecules, inorganic compounds, nanoparticles,enzymes or enzyme substrates, fluorescent materials, luminescentmaterials (e.g., luminol), bioluminescent materials (e.g., luciferase,luciferin, and aequorin), chemiluminescent materials, radioactivematerials (e.g., ¹⁸F, ⁶⁷ Ga, ^(81m)Kr, ⁸²Rb, ¹¹¹In, ¹²³I, ¹³³Xe, ²⁰¹Tl,¹²⁵I, ³⁵S, ¹⁴C, ³H, or ^(99m)Tc (e.g., as pertechnetate(technetate(VII), TcO₄ ⁻ )), and contrast agents (e.g., gold (e.g., goldnanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g.,superparamagnetic iron oxide (SPIO), monocrystalline iron oxidenanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide(USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinatedcontrast media (iohexol), microbubbles, or perfluorocarbons). Suchoptically-detectable labels include for example, without limitation,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives (e.g., acridine and acridine isothiocyanate);5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives (e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120), and7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes;cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI); 5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives (e.g., eosin and eosin isothiocyanate); erythrosin andderivatives (e.g., erythrosin B and erythrosin isothiocyanate);ethidium; fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITCor XRITC), and fluorescamine);2-[2-[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benz[e]indol-2-ylidene]ethylidene]-2-[4-(ethoxycarbonyl)-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulforpropyl)-1H-benz[e]indoliumhydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144);5-chloro-2-[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazol-ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140); Malachite Green isothiocyanate;4-methylumbelliferone orthocresolphthalein; nitrotyrosine;pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyreneand derivatives(e.g., pyrene, pyrene butyrate, and succinimidyl1-pyrene); butyrate quantum dots; Reactive Red 4 (CIBACRON™ BrilliantRed 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine Xisothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloridederivative of sulforhodamine 101 (Texas Red), N,N,N′,N′tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine, andtetramethyl rhodamine isothiocyanate (TRITC)); riboflavin; rosolic acid;terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; LaJolta Blue; phthalo cyanine; and naphthalo cyanine.

In some embodiments, the detectable agent may be a non-detectablepre-cursor that becomes detectable upon activation (e.g., fluorogenictetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL,tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzymeactivatable fluorogenic agents (e.g., PROSENSE® (VisEn Medical))). Invitro assays in which the enzyme labeled compositions can be usedinclude, but are not limited to, enzyme linked immunosorbent assays(ELISAs), immunoprecipitation assays, immunofluorescence, enzymeimmunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.Combinations

The polynucleotides, primary constructs or mmRNA may be used incombination with one or more other therapeutic, prophylactic,diagnostic, or imaging agents. By “in combination with,” it is notintended to imply that the agents must be administered at the same timeand/or formulated for delivery together, although these methods ofdelivery are within the scope of the present disclosure. Compositionscan be administered concurrently with, prior to, or subsequent to, oneor more other desired therapeutics or medical procedures. In general,each agent will be administered at a dose and/or on a time scheduledetermined for that agent. In some embodiments, the present disclosureencompasses the delivery of pharmaceutical, prophylactic, diagnostic, orimaging compositions in combination with agents that may improve theirbioavailability, reduce and/or modify their metabolism, inhibit theirexcretion, and/or modify their distribution within the body. As anon-limiting example, the nucleic acids or mmRNA may be used incombination with a pharmaceutical agent for the treatment of cancer orto control hyperproliferative cells. In U.S. Pat. No. 7,964,571, hereinincorporated by reference in its entirety, a combination therapy for thetreatment of solid primary or metastasized tumor is described using apharmaceutical composition including a DNA plasmid encoding forinterleukin-12 with a lipopolymer and also administering at least oneanticancer agent or chemotherapeutic. Further, the nucleic acids andmmRNA of the present invention that encodes anti-proliferative moleculesmay be in a pharmaceutical composition with a lipopolymer (see e.g.,U.S. Pub. No. 20110218231, herein incorporated by reference in itsentirety, claiming a pharmaceutical composition comprising a DNA plasmidencoding an anti-proliferative molecule and a lipopolymer) which may beadministered with at least one chemotherapeutic or anticancer agent.

Dosing

The present invention provides methods comprising administering modifiedmRNAs and their encoded proteins or complexes in accordance with theinvention to a subject in need thereof. Nucleic acids, proteins orcomplexes, or pharmaceutical, imaging, diagnostic, or prophylacticcompositions thereof, may be administered to a subject using any amountand any route of administration effective for preventing, treating,diagnosing, or imaging a disease, disorder, and/or condition (e.g., adisease, disorder, and/or condition relating to working memorydeficits). The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Compositions inaccordance with the invention are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositions ofthe present invention may be decided by the attending physician withinthe scope of sound medical judgment. The specific therapeuticallyeffective, prophylactically effective, or appropriate imaging dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

In certain embodiments, compositions in accordance with the presentinvention may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg toabout 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg toabout 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect. The desired dosage may be deliveredthree times a day, two times a day, once a day, every other day, everythird day, every week, every two weeks, every three weeks, or every fourweeks. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations).

According to the present invention, it has been discovered thatadministration of mmRNA in split-dose regimens produce higher levels ofproteins in mammalian subjects. As used herein, a “split dose” is thedivision of single unit dose or total daily dose into two or more doses,e.g, two or more administrations of the single unit dose. As usedherein, a “single unit dose” is a dose of any therapeutic administed inone dose/at one time/single route/single point of contact, i.e., singleadministration event. As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose. In one embodiment, the mmRNA of the present invention areadministed to a subject in split doses. The mmRNA may be formulated inbuffer only or in a formulation described herein.

Dosage Forms

A pharmaceutical composition described herein can be formulated into adosage form described herein, such as a topical, intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intracardiac, intraperitoneal,subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration include, but are notlimited to, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and/or elixirs. In addition to activeingredients, liquid dosage forms may comprise inert diluents commonlyused in the art including, but not limited to, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. In certainembodiments for parenteral administration, compositions may be mixedwith solubilizing agents such as CREMOPHOR®, alcohols, oils, modifiedoils, glycols, polysorbates, cyclodextrins, polymers, and/orcombinations thereof.

Injectable

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known art andmay include suitable dispersing agents, wetting agents, and/orsuspending agents. Sterile injectable preparations may be sterileinjectable solutions, suspensions, and/or emulsions in nontoxicparenterally acceptable diluents and/or solvents, for example, asolution in 1,3-butanediol. Among the acceptable vehicles and solventsthat may be employed include, but are not limited to, are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution.Sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil can be employedincluding synthetic mono- or diglycerides. Fatty acids such as oleicacid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it may bedesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the polynucleotide,primary construct or mmRNA then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administeredpolynucleotide, primary construct or mmRNA may be accomplished bydissolving or suspending the polynucleotide, primary construct or mmRNAin an oil vehicle. Injectable depot forms are made by formingmicroencapsule matrices of the polynucleotide, primary construct ormmRNA in biodegradable polymers such as polylactide-polyglycolide.Depending upon the ratio of polynucleotide, primary construct or mmRNAto polymer and the nature of the particular polymer employed, the rateof polynucleotide, primary construct or mmRNA release can be controlled.Examples of other biodegradable polymers include, but are not limitedto, poly(orthoesters) and poly(anhydrides). Depot injectableformulations may be prepared by entrapping the polynucleotide, primaryconstruct or mmRNA in liposomes or microemulsions which are compatiblewith body tissues.

Pulmonary

Formulations described herein as being useful for pulmonary delivery mayalso be use for intranasal delivery of a pharmaceutical composition.Another formulation suitable for intranasal administration may be acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 μm to 500 μm. Such a formulation may beadministered in the manner in which snuff is taken, i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, contain about 0.1% to 20% (w/w) active ingredient, where thebalance may comprise an orally dissolvable and/or degradable compositionand, optionally, one or more of the additional ingredients describedherein. Alternately, formulations suitable for buccal administration maycomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising active ingredient. Such powdered, aerosolized,and/or aerosolized formulations, when dispersed, may have an averageparticle and/or droplet size in the range from about 0.1 nm to about 200nm, and may further comprise one or more of any additional ingredientsdescribed herein.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granulescan be prepared with coatings and shells such as enteric coatings andother coatings well known in the pharmaceutical formulating art. Theymay optionally comprise opacifying agents and can be of a compositionthat they release the active ingredient(s) only, or preferentially, in acertain part of the intestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. Solid compositions of a similar type may beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like.

Properties of Pharmaceutical Compositions

The pharmaceutical compositions described herein can be characterized byone or more of bioavailability, therapeutic window and/or volume ofdistribution.

Bioavailability

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit anincrease in bioavailability as compared to a composition lacking adelivery agent as described herein. As used herein, the term“bioavailability” refers to the systemic availability of a given amountof polynucleotides, primary constructs or mmRNA administered to amammal. Bioavailability can be assessed by measuring the area under thecurve (AUC) or the maximum serum or plasma concentration (C_(max)) ofthe unchanged form of a compound following administration of thecompound to a mammal. AUC is a determination of the area under the curveplotting the serum or plasma concentration of a compound along theordinate (Y-axis) against time along the abscissa (X-axis). Generally,the AUC for a particular compound can be calculated using methods knownto those of ordinary skill in the art and as described in G. S. Banker,Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72,Marcel Dekker, New York, Inc., 1996, herein incorporated by reference.

The C_(max) value is the maximum concentration of the compound achievedin the serum or plasma of a mammal following administration of thecompound to the mammal. The C_(max) value of a particular compound canbe measured using methods known to those of ordinary skill in the art.The phrases “increasing bioavailability” or “improving thepharmacokinetics,” as used herein mean that the systemic availability ofa first polynucleotide, primary construct or mmRNA, measured as AUC,C_(max), or C_(min) in a mammal is greater, when co-administered with adelivery agent as described herein, than when such co-administrationdoes not take place. In some embodiments, the bioavailability of thepolynucleotide, primary construct or mmRNA can increase by at leastabout 2%, at least about 5%, at least about 10%, at least about 15%, atleast about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or about 100%.

Therapeutic Window

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit anincrease in the therapeutic window of the administered polynucleotide,primary construct or mmRNA composition as compared to the therapeuticwindow of the administered polynucleotide, primary construct or mmRNAcomposition lacking a delivery agent as described herein. As used herein“therapeutic window” refers to the range of plasma concentrations, orthe range of levels of therapeutically active substance at the site ofaction, with a high probability of eliciting a therapeutic effect. Insome embodiments, the therapeutic window of the polynucleotide, primaryconstruct or mmRNA when co-administered with a delivery agent asdescribed herein can increase by at least about 2%, at least about 5%,at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100%.

Volume of Distribution

The polynucleotides, primary constructs or mmRNA, when formulated into acomposition with a delivery agent as described herein, can exhibit animproved volume of distribution (V_(dist)), e.g., reduced or targeted,relative to a composition lacking a delivery agent as described herein.The volume of distribution (V_(dist)) relates the amount of the drug inthe body to the concentration of the drug in the blood or plasma. Asused herein, the term “volume of distribution” refers to the fluidvolume that would be required to contain the total amount of the drug inthe body at the same concentration as in the blood or plasma: V_(dist)equals the amount of drug in the body/concentration of drug in blood orplasma. For example, for a 10 mg dose and a plasma concentration of 10mg/L, the volume of distribution would be 1 liter. The volume ofdistribution reflects the extent to which the drug is present in theextravascular tissue. A large volume of distribution reflects thetendency of a compound to bind to the tissue components compared withplasma protein binding. In a clinical setting, V_(dist) can be used todetermine a loading dose to achieve a steady state concentration. Insome embodiments, the volume of distribution of the polynucleotide,primary construct or mmRNA when co-administered with a delivery agent asdescribed herein can decrease at least about 2%, at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%.

Biological Effect

In one embodiment, the biological effect of the modified mRNA deliveredto the animals may be categorized by analyzing the protein expression inthe animals. The protein expression may be determined from analyzing abiological sample collected from a mammal administered the modified mRNAof the present invention. In one embodiment, the expression proteinencoded by the modified mRNA administered to the mammal of at least 50pg/ml may be preferred. For example, a protein expression of 50-200pg/ml for the protein encoded by the modified mRNA delivered to themammal may be seen as a therapeutically effective amount of protein inthe mammal.

Detection of Modified Nucleic Acids by Mass Spectrometry

Mass spectrometry (MS) is an analytical technique that can providestructural and molecular mass/concentration information on moleculesafter their conversion to ions. The molecules are first ionized toacquire positive or negative charges and then they travel through themass analyzer to arrive at different areas of the detector according totheir mass/charge (m/z) ratio.

Mass spectrometry is performed using a mass spectrometer which includesan ion source for ionizing the fractionated sample and creating chargedmolecules for further analysis. For example ionization of the sample maybe performed by electrospray ionization (ESI), atmospheric pressurechemical ionization (APCI), photoionization, electron ionization, fastatom bombardment (FAB)/liquid secondary ionization (LSIMS), matrixassisted laser desorption/ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, and particle beamionization. The skilled artisan will understand that the choice ofionization method can be determined based on the analyte to be measured,type of sample, the type of detector, the choice of positive versusnegative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Suitable analyzers for determiningmass-to-charge ratios include quadropole analyzers, ion traps analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM).

Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupledwith stable isotope labeled dilution of peptide standards has been shownto be an effective method for protein verification (e.g., Keshishian etal., Mol Cell Proteomics 2009 8: 2339-2349; Kuhn et al., Clin Chem 200955:1108-1117; Lopez et al., Clin Chem 2010 56:281-290). Unlikeuntargeted mass spectrometry frequently used in biomarker discoverystudies, targeted MS methods are peptide sequence-based modes of MS thatfocus the full analytical capacity of the instrument on tens to hundredsof selected peptides in a complex mixture. By restricting detection andfragmentation to only those peptides derived from proteins of interest,sensitivity and reproducibility are improved dramatically compared todiscovery-mode MS methods. This method of mass spectrometry-basedmultiple reaction monitoring (MRM) quantitation of proteins candramatically impact the discovery and quantitation of biomarkers viarapid, targeted, multiplexed protein expression profiling of clinicalsamples.

In one embodiment, a biological sample which may contain at least oneprotein encoded by at least one modified mRNA of the present inventionmay be analyzed by the method of MRM-MS. The quantification of thebiological sample may further include, but is not limited to,isotopically labeled peptides or proteins as internal standards.

According to the present invention, the biological sample, once obtainedfrom the subject, may be subjected to enzyme digestion. As used herein,the term “digest” means to break apart into shorter peptides. As usedherein, the phrase “treating a sample to digest proteins” meansmanipulating a sample in such a way as to break down proteins in asample. These enzymes include, but are not limited to, trypsin,endoproteinase Glu-C and chymotrypsin. In one embodiment, a biologicalsample which may contain at least one protein encoded by at least onemodified mRNA of the present invention may be digested using enzymes.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for proteinusing electrospray ionization. Electrospray ionization (ESI) massspectrometry (ESIMS) uses electrical energy to aid in the transfer ofions from the solution to the gaseous phase before they are analyzed bymass spectrometry. Samples may be analyzed using methods known in theart (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12). The ionicspecies contained in solution may be transferred into the gas phase bydispersing a fine spray of charge droplets, evaporating the solvent andejecting the ions from the charged droplets to generate a mist of highlycharged droplets. The mist of highly charged droplets may be analyzedusing at least 1, at least 2, at least 3 or at least 4 mass analyzerssuch as, but not limited to, a quadropole mass analyzer. Further, themass spectrometry method may include a purification step. As anon-limiting example, the first quadrapole may be set to select a singlem/z ratio so it may filter out other molecular ions having a differentm/z ratio which may eliminate complicated and time-consuming samplepurification procedures prior to MS analysis.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed for protein ina tandem ESIMS system (e.g., MS/MS). As non-limiting examples, thedroplets may be analyzed using a product scan (or daughter scan) aprecursor scan (parent scan) a neutral loss or a multiple reactionmonitoring.

In one embodiment, a biological sample which may contain protein encodedby modified mRNA of the present invention may be analyzed usingmatrix-assisted laser desorption/ionization (MALDI) mass spectrometry(MALDIMS). MALDI provides for the nondestructive vaporization andionization of both large and small molecules, such as proteins. In MALDIanalysis, the analyte is first co-crystallized with a large molar excessof a matrix compound, which may also include, but is not limited to, anultraviolet absorbing weak organic acid. Non-limiting examples ofmatrices used in MALDI are α-cyano-4-hydroxycinnamic acid,3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.Laser radiation of the analyte-matrix mixture may result in thevaporization of the matrix and the analyte. The laser induced desorptionprovides high ion yields of the intact analyte and allows formeasurement of compounds with high accuracy. Samples may be analyzedusing methods known in the art (e.g., Lewis, Wei and Siuzdak,Encyclopedia of Analytical Chemistry 2000:5880-5894). As non-limitingexamples, mass analyzers used in the MALDI analysis may include a lineartime-of-flight (TOF), a TOF reflectron or a Fourier transform massanalyzer.

In one embodiment, the analyte-matrix mixture may be formed using thedried-droplet method. A biologic sample is mixed with a matrix to createa saturated matrix solution where the matrix-to-sample ratio isapproximately 5000:1. An aliquot (approximately 0.5-2.0 uL) of thesaturated matrix solution is then allowed to dry to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethin-layer method. A matrix homogeneous film is first formed and thenthe sample is then applied and may be absorbed by the matrix to form theanalyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thethick-layer method. A matrix homogeneous film is formed with anitro-cellulose matrix additive. Once the uniform nitro-cellulose matrixlayer is obtained the sample is applied and absorbed into the matrix toform the analyte-matrix mixture.

In one embodiment, the analyte-matrix mixture may be formed using thesandwich method. A thin layer of matrix crystals is prepared as in thethin-layer method followed by the addition of droplets of aqueoustrifluoroacetic acid, the sample and matrix. The sample is then absorbedinto the matrix to form the analyte-matrix mixture.

V. USES OF POLYNUCLEOTIDES, PRIMARY CONSTRUCTS AND MMRNA OF THEINVENTION

The polynucleotides, primary constructs and mmRNA of the presentinvention are designed, in preferred embodiments, to provide foravoidance or evasion of deleterious bio-responses such as the immuneresponse and/or degradation pathways, overcoming the threshold ofexpression and/or improving protein production capacity, improvedexpression rates or translation efficiency, improved drug or proteinhalf life and/or protein concentrations, optimized protein localization,to improve one or more of the stability and/or clearance in tissues,receptor uptake and/or kinetics, cellular access by the compositions,engagement with translational machinery, secretion efficiency (whenapplicable), accessibility to circulation, and/or modulation of a cell'sstatus, function and/or activity.

Therapeutics Therapeutic Agents

The polynucleotides, primary constructs or mmRNA of the presentinvention, such as modified nucleic acids and modified RNAs, and theproteins translated from them described herein can be used astherapeutic or prophylactic agents. They are provided for use inmedicine. For example, a polynucleotide, primary construct or mmRNAdescribed herein can be administered to a subject, wherein thepolynucleotide, primary construct or mmRNA is translated in vivo toproduce a therapeutic or prophylactic polypeptide in the subject.Provided are compositions, methods, kits, and reagents for diagnosis,treatment or prevention of a disease or condition in humans and othermammals. The active therapeutic agents of the invention includepolynucleotides, primary constructs or mmRNA, cells containingpolynucleotides, primary constructs or mmRNA or polypeptides translatedfrom the polynucleotides, primary constructs or mmRNA.

In certain embodiments, provided herein are combination therapeuticscontaining one or more polynucleotide, primary construct or mmRNAcontaining translatable regions that encode for a protein or proteinsthat boost a mammalian subject's immunity along with a protein thatinduces antibody-dependent cellular toxicity. For example, providedherein are therapeutics containing one or more nucleic acids that encodetrastuzumab and granulocyte-colony stimulating factor (G-CSF). Inparticular, such combination therapeutics are useful in Her2+ breastcancer patients who develop induced resistance to trastuzumab. (See,e.g., Albrecht, Immunotherapy. 2(6):795-8 (2010)).

Provided herein are methods of inducing translation of a recombinantpolypeptide in a cell population using the polynucleotide, primaryconstruct or mmRNA described herein. Such translation can be in vivo, exvivo, in culture, or in vitro. The cell population is contacted with aneffective amount of a composition containing a nucleic acid that has atleast one nucleoside modification, and a translatable region encodingthe recombinant polypeptide. The population is contacted underconditions such that the nucleic acid is localized into one or morecells of the cell population and the recombinant polypeptide istranslated in the cell from the nucleic acid.

An “effective amount” of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the nucleic acid (e.g., size, and extent ofmodified nucleosides), and other determinants. In general, an effectiveamount of the composition provides efficient protein production in thecell, preferably more efficient than a composition containing acorresponding unmodified nucleic acid. Increased efficiency may bedemonstrated by increased cell transfection (i.e., the percentage ofcells transfected with the nucleic acid), increased protein translationfrom the nucleic acid, decreased nucleic acid degradation (asdemonstrated, e.g., by increased duration of protein translation from amodified nucleic acid), or reduced innate immune response of the hostcell.

Aspects of the invention are directed to methods of inducing in vivotranslation of a recombinant polypeptide in a mammalian subject in needthereof. Therein, an effective amount of a composition containing anucleic acid that has at least one structural or chemical modificationand a translatable region encoding the recombinant polypeptide isadministered to the subject using the delivery methods described herein.The nucleic acid is provided in an amount and under other conditionssuch that the nucleic acid is localized into a cell of the subject andthe recombinant polypeptide is translated in the cell from the nucleicacid. The cell in which the nucleic acid is localized, or the tissue inwhich the cell is present, may be targeted with one or more than onerounds of nucleic acid administration.

In certain embodiments, the administered polynucleotide, primaryconstruct or mmRNA directs production of one or more recombinantpolypeptides that provide a functional activity which is substantiallyabsent in the cell, tissue or organism in which the recombinantpolypeptide is translated. For example, the missing functional activitymay be enzymatic, structural, or gene regulatory in nature. In relatedembodiments, the administered polynucleotide, primary construct or mmRNAdirects production of one or more recombinant polypeptides thatincreases (e.g., synergistically) a functional activity which is presentbut substantially deficient in the cell in which the recombinantpolypeptide is translated.

In other embodiments, the administered polynucleotide, primary constructor mmRNA directs production of one or more recombinant polypeptides thatreplace a polypeptide (or multiple polypeptides) that is substantiallyabsent in the cell in which the recombinant polypeptide is translated.Such absence may be due to genetic mutation of the encoding gene orregulatory pathway thereof. In some embodiments, the recombinantpolypeptide increases the level of an endogenous protein in the cell toa desirable level; such an increase may bring the level of theendogenous protein from a subnormal level to a normal level or from anormal level to a super-normal level.

Alternatively, the recombinant polypeptide functions to antagonize theactivity of an endogenous protein present in, on the surface of, orsecreted from the cell. Usually, the activity of the endogenous proteinis deleterious to the subject; for example, due to mutation of theendogenous protein resulting in altered activity or localization.Additionally, the recombinant polypeptide antagonizes, directly orindirectly, the activity of a biological moiety present in, on thesurface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a nucleic acid, a carbohydrate, aprotein toxin such as shiga and tetanus toxins, or a small moleculetoxin such as botulinum, cholera, and diphtheria toxins. Additionally,the antagonized biological molecule may be an endogenous protein thatexhibits an undesirable activity, such as a cytotoxic or cytostaticactivity.

The recombinant proteins described herein may be engineered forlocalization within the cell, potentially within a specific compartmentsuch as the nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

In some embodiments, modified mRNAs and their encoded polypeptides inaccordance with the present invention may be used for treatment of anyof a variety of diseases, disorders, and/or conditions, including butnot limited to one or more of the following: autoimmune disorders (e.g.diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis);inflammatory disorders (e.g. arthritis, pelvic inflammatory disease);infectious diseases (e.g. viral infections (e.g., HIV, HCV, RSV),bacterial infections, fungal infections, sepsis); neurological disorders(e g. Alzheimer's disease, Huntington's disease; autism; Duchennemuscular dystrophy); cardiovascular disorders (e.g. atherosclerosis,hypercholesterolemia, thrombosis, clotting disorders, angiogenicdisorders such as macular degeneration); proliferative disorders (e.g.cancer, benign neoplasms); respiratory disorders (e.g. chronicobstructive pulmonary disease); digestive disorders (e.g. inflammatorybowel disease, ulcers); musculoskeletal disorders (e.g. fibromyalgia,arthritis); endocrine, metabolic, and nutritional disorders (e.g.diabetes, osteoporosis); urological disorders (e.g. renal disease);psychological disorders (e.g. depression, schizophrenia); skin disorders(e.g. wounds, eczema); blood and lymphatic disorders (e.g. anemia,hemophilia); etc.

Diseases characterized by dysfunctional or aberrant protein activityinclude cystic fibrosis, sickle cell anemia, epidermolysis bullosa,amyotrophic lateral sclerosis, and glucose-6-phosphate dehydrogenasedeficiency. The present invention provides a method for treating suchconditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the polynucleotide, primary constructor mmRNA provided herein, wherein the polynucleotide, primary constructor mmRNA encode for a protein that antagonizes or otherwise overcomesthe aberrant protein activity present in the cell of the subject.Specific examples of a dysfunctional protein are the missense mutationvariants of the cystic fibrosis transmembrane conductance regulator(CFTR) gene, which produce a dysfunctional protein variant of CFTRprotein, which causes cystic fibrosis.

Diseases characterized by missing (or substantially diminished such thatproper (normal or physiological protein function does not occur) proteinactivity include cystic fibrosis, Niemann-Pick type C, β thalassemiamajor, Duchenne muscular dystrophy, Hurler Syndrome, Hunter Syndrome,and Hemophilia A. Such proteins may not be present, or are essentiallynon-functional. The present invention provides a method for treatingsuch conditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the polynucleotide, primary constructor mmRNA provided herein, wherein the polynucleotide, primary constructor mmRNA encode for a protein that replaces the protein activity missingfrom the target cells of the subject. Specific examples of adysfunctional protein are the nonsense mutation variants of the cysticfibrosis transmembrane conductance regulator (CFTR) gene, which producea nonfunctional protein variant of CFTR protein, which causes cysticfibrosis.

Thus, provided are methods of treating cystic fibrosis in a mammaliansubject by contacting a cell of the subject with a polynucleotide,primary construct or mmRNA having a translatable region that encodes afunctional CFTR polypeptide, under conditions such that an effectiveamount of the CTFR polypeptide is present in the cell. Preferred targetcells are epithelial, endothelial and mesothelial cells, such as thelung, and methods of administration are determined in view of the targettissue; i.e., for lung delivery, the RNA molecules are formulated foradministration by inhalation.

In another embodiment, the present invention provides a method fortreating hyperlipidemia in a subject, by introducing into a cellpopulation of the subject with a modified mRNA molecule encodingSortilin, a protein recently characterized by genomic studies, therebyameliorating the hyperlipidemia in a subject. The SORT1 gene encodes atrans-Golgi network (TGN) transmembrane protein called Sortilin. Geneticstudies have shown that one of five individuals has a single nucleotidepolymorphism, rs12740374, in the 1p13 locus of the SORT1 gene thatpredisposes them to having low levels of low-density lipoprotein (LDL)and very-low-density lipoprotein (VLDL). Each copy of the minor allele,present in about 30% of people, alters LDL cholesterol by 8 mg/dL, whiletwo copies of the minor allele, present in about 5% of the population,lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have alsobeen shown to have a 40% decreased risk of myocardial infarction.Functional in vivo studies in mice describes that overexpression ofSORT1 in mouse liver tissue led to significantly lower LDL-cholesterollevels, as much as 80% lower, and that silencing SORT1 increased LDLcholesterol approximately 200% (Musunuru K et al. From noncoding variantto phenotype via SORT1 at the 1p13 cholesterol locus. Nature 2010; 466:714-721).

Provided herein, are methods to prevent infection and/or sepsis in asubject at risk of developing infection and/or sepsis, the methodcomprising administering to a subject in need of such prevention acomposition comprising a polynucleotide, primary construct or mmRNAprecursor encoding an anti-microbial polypeptide (e.g., ananti-bacterial polypeptide), or a partially or fully processed formthereof in an amount sufficient to prevent infection and/or sepsis. Incertain embodiments, the subject at risk of developing infection and/orsepsis may be a cancer patient. In certain embodiments, the cancerpatient may have undergone a conditioning regimen. In some embodiments,the conditioning regiment may include, but is not limited to,chemotherapy, radiation therapy, or both.

Further provided herein, are methods to treat infection and/or sepsis ina subject, the method comprising administering to a subject in need ofsuch treatment a composition comprising a polynucleotide, primaryconstruct or mmRNA precursor encoding an anti-microbial polypeptide(e.g., an anti-bacterial polypeptide), e.g., an anti-microbialpolypeptide described herein, or a partially or fully processed formthereof in an amount sufficient to treat an infection and/or sepsis. Incertain embodiments, the subject in need of treatment is a cancerpatient. In certain embodiments, the cancer patient has undergone aconditioning regimen. In some embodiments, the conditioning regiment mayinclude, but is not limited to, chemotherapy, radiation therapy, orboth.

In certain embodiments, the subject may exhibits acute or chronicmicrobial infections (e.g., bacterial infections). In certainembodiments, the subject may have received or may be receiving atherapy. In certain embodiments, the therapy may include, but is notlimited to, radiotherapy, chemotherapy, steroids, ultraviolet radiation,or a combination thereof. In certain embodiments, the patient may sufferfrom a microvascular disorder. In some embodiments, the microvasculardisorder may be diabetes. In certain embodiments, the patient may have awound. In some embodiments, the wound may be an ulcer. In a specificembodiment, the wound may be a diabetic foot ulcer. In certainembodiments, the subject may have one or more burn wounds. In certainembodiments, the administration may be local or systemic. In certainembodiments, the administration may be subcutaneous. In certainembodiments, the administration may be intravenous. In certainembodiments, the administration may be oral. In certain embodiments, theadministration may be topical. In certain embodiments, theadministration may be by inhalation. In certain embodiments, theadministration may be rectal. In certain embodiments, the administrationmay be vaginal.

Other aspects of the present disclosure relate to transplantation ofcells containing polynucleotide, primary construct, or mmRNA to amammalian subject. Administration of cells to mammalian subjects isknown to those of ordinary skill in the art, and include, but is notlimited to, local implantation (e.g., topical or subcutaneousadministration), organ delivery or systemic injection (e.g., intravenousinjection or inhalation), and the formulation of cells inpharmaceutically acceptable carrier. Such compositions containingpolynucleotide, primary construct, or mmRNA can be formulated foradministration intramuscularly, transarterially, intraperitoneally,intravenously, intranasally, subcutaneously, endoscopically,transdermally, or intrathecally. In some embodiments, the compositionmay be formulated for extended release.

The subject to whom the therapeutic agent may be administered suffersfrom or may be at risk of developing a disease, disorder, or deleteriouscondition. Provided are methods of identifying, diagnosing, andclassifying subjects on these bases, which may include clinicaldiagnosis, biomarker levels, genome-wide association studies (GWAS), andother methods known in the art.

Bioprocessing

The methods provided herein may be useful for enhancing protein productyield in a cell culture process. Specifically, the novel enzymes andenzyme variants identified which are useful in the preparation ofchemically modified mRNA may also be used in bioprocessing reactions. Ina cell culture containing a plurality of host cells, introduction of apolynucleotide, primary construct or mmRNA described herein results inincreased protein production efficiency relative to a correspondingunmodified nucleic acid. Such increased protein production efficiencycan be demonstrated, e.g., by showing increased cell transfection,increased protein translation from the polynucleotide, primary constructor mmRNA, decreased nucleic acid degradation, and/or reduced innateimmune response of the host cell. Protein production can be measured byenzyme-linked immunosorbent assay (ELISA), and protein activity can bemeasured by various functional assays known in the art. The proteinproduction may be generated in a continuous or a batch-fed mammalianprocess.

Additionally, it is useful to optimize the expression of a specificpolypeptide in a cell line or collection of cell lines of potentialinterest, particularly a polypeptide of interest such as a proteinvariant of a reference protein having a known activity. In oneembodiment, provided is a method of optimizing expression of apolypeptide of interest in a target cell, by providing a plurality oftarget cell types, and independently contacting with each of theplurality of target cell types a polynucleotide, primary construct ormmRNA encoding a polypeptide of interest. The cells may be transfectedwith two or more polynucleotide, primary construct or mmRNAsimultaneously or sequentially.

In certain embodiments, multiple rounds of the methods described hereinmay be used to obtain cells with increased expression of one or morenucleic acids or proteins of interest. For example, cells may betransfected with one or more polynucleotide, primary construct or mmRNAthat encode a nucleic acid or protein of interest. The cells may beisolated according to methods described herein before being subjected tofurther rounds of transfections with one or more other nucleic acidswhich encode a nucleic acid or protein of interest before being isolatedagain. This method may be useful for generating cells with increasedexpression of a complex of proteins, nucleic acids or proteins in thesame or related biological pathway, nucleic acids or proteins that actupstream or downstream of each other, nucleic acids or proteins thathave a modulating, activating or repressing function to each other,nucleic acids or proteins that are dependent on each other for functionor activity, or nucleic acids or proteins that share homology.

Additionally, culture conditions may be altered to increase proteinproduction efficiency. Subsequently, the presence and/or level of thepolypeptide of interest in the plurality of target cell types isdetected and/or quantitated, allowing for the optimization of apolypeptide's expression by selection of an efficient target cell andcell culture conditions relating thereto. Such methods are particularlyuseful when the polypeptide contains one or more post-translationalmodifications or has substantial tertiary structure, situations whichoften complicate efficient protein production.

In one embodiment, the cells used in the methods of the presentinvention may be cultured. The cells may be cultured in suspension or asadherent cultures. The cells may be cultured in a varied of vesselsincluding, but not limited to, bioreactors, cell bags, wave bags,culture plates, flasks and other vessels well known to those of ordinaryskill in the art. Cells may be cultured in IMDM (Invitrogen, Catalognumber 12440-53) or any other suitable media including, but not limitedto, chemically defined media formulations. The ambient conditions whichmay be suitable for cell culture, such as temperature and atmosphericcomposition, are well known to those skilled in the art. The methods ofthe invention may be used with any cell that is suitable for use inprotein production.

The invention provides for the repeated introduction (e.g.,transfection) of modified nucleic acids into a target cell population,e.g., in vitro, ex vivo, in situ, or in vivo. For example, contactingthe same cell population may be repeated one or more times (such as two,three, four, five or more than five times). In some embodiments, thestep of contacting the cell population with the polynucleotides, primaryconstructs or mmRNA is repeated a number of times sufficient such that apredetermined efficiency of protein translation in the cell populationis achieved. Given the often reduced cytotoxicity of the target cellpopulation provided by the nucleic acid modifications, repeatedtransfections are achievable in a diverse array of cell types and withina variety of tissues, as provided herein.

In one embodiment, the bioprocessing methods of the present inventionmay be used to produce antibodies or functional fragments thereof. Thefunctional fragments may comprise a Fab, Fab′, F(ab′)₂, an Fv domain, anscFv, or a diabody. They may be variable in any region including thecomplement determining region (CDR). In one embodiment, there iscomplete diversity in the CDR3 region. In another embodiment, theantibody is substantially conserved except in the CDR3 region.

Antibodies may be made which bind or associate with any biomolecule,whether human, pathogenic or non-human in origin. The pathogen may bepresent in a non-human mammal, a clinical specimen or from a commercialproduct such as a cosmetic or pharmaceutical material. They may alsobind to any specimen or sample including clinical specimens or tissuesamples from any organism.

In some embodiments, the contacting step is repeated multiple times at afrequency selected from the group consisting of: 6 hr, 12 hr, 24 hr, 36hr, 48 hr, 72 hr, 84 hr, 96 hr, and 108 hr and at concentrations of lessthan 20 nM, less than 50 nM, less than 80 nM or less than 100 nM.Compositions may also be administered at less than 1 mM, less than 5 mM,less than 10 mM, less than 100 mM or less than 500 mM.

In some embodiments, the polynucleotides, primary constructs or mmRNAare added at an amount of 50 molecules per cell, 100 molecules/cell, 200molecules/cell, 300 molecules/cell, 400 molecules/cell, 500molecules/cell, 600 molecules/cell, 700 molecules/cell, 800molecules/cell, 900 molecules/cell, 1000 molecules/cell, 2000molecules/cell, or 5000 molecules/cell.

In other embodiments, the polynucleotides, primary constructs or mmRNAare added at a concentration selected from the group consisting of: 0.01fmol/106 cells, 0.1 fmol/106 cells, 0.5 fmol/106 cells, 0.75 fmol/106cells, 1 fmol/106 cells, 2 fmol/106 cells, 5 fmol/106 cells, 10 fmol/106cells, 20 fmol/106 cells, 30 fmol/106 cells, 40 fmol/106 cells, 50fmol/106 cells, 60 fmol/106 cells, 100 fmol/106 cells, 200 fmol/106cells, 300 fmol/106 cells, 400 fmol/106 cells, 500 fmol/106 cells, 700fmol/106 cells, 800 fmol/106 cells, 900 fmol/106 cells, and 1 pmol/106cells.

In some embodiments, the production of a biological product upon isdetected by monitoring one or more measurable bioprocess parameters,such as a parameter selected from the group consisting of: cell density,pH, oxygen levels, glucose levels, lactic acid levels, temperature, andprotein production. Protein production can be measured as specificproductivity (SP) (the concentration of a product, such as aheterologously expressed polypeptide, in solution) and can be expressedas mg/L or g/L; in the alternative, specific productivity can beexpressed as pg/cell/day. An increase in SP can refer to an absolute orrelative increase in the concentration of a product produced under twodefined set of conditions (e.g., when compared with controls not treatedwith modified mRNA(s)).

Cells

In one embodiment, the cells are selected from the group consisting ofmammalian cells, bacterial cells, plant, microbial, algal and fungalcells. In some embodiments, the cells are mammalian cells, such as, butnot limited to, human, mouse, rat, goat, horse, rabbit, hamster or cowcells. In a further embodiment, the cells may be from an establishedcell line, including, but not limited to, HeLa, NSO, SP2/0, KEK 293T,Vero, Caco, Caco-2, MDCK, COS-1, COS-7, K562, Jurkat, CHO-K1, DG44,CHOK1SV, CHO-S, Huvec, CV-1, Huh-7, NIH3T3, HEK293, 293, A549, HepG2,IMR-90, MCF-7, U-20S, Per.C6, SF9, SF21 or Chinese Hamster Ovary (CHO)cells.

In certain embodiments, the cells are fungal cells, such as, but notlimited to, Chrysosporium cells, Aspergillus cells, Trichoderma cells,Dictyostelium cells, Candida cells, Saccharomyces cells,Schizosaccharomyces cells, and Penicillium cells.

In certain embodiments, the cells are bacterial cells such as, but notlimited to, E. coli, B. subtilis, or BL21 cells. Primary and secondarycells to be transfected by the methods of the invention can be obtainedfrom a variety of tissues and include, but are not limited to, all celltypes which can be maintained in culture. For examples, primary andsecondary cells which can be transfected by the methods of the inventioninclude, but are not limited to, fibroblasts, keratinocytes, epithelialcells (e.g., mammary epithelial cells, intestinal epithelial cells),endothelial cells, glial cells, neural cells, formed elements of theblood (e.g., lymphocytes, bone marrow cells), muscle cells andprecursors of these somatic cell types. Primary cells may also beobtained from a donor of the same species or from another species (e.g.,mouse, rat, rabbit, cat, dog, pig, cow, bird, sheep, goat, horse).

Purification and Isolation

Those of ordinary skill in the art should be able to make adetermination of the methods to use to purify or isolate of a protein ofinterest from cultured cells. Generally, this is done through a capturemethod using affinity binding or non-affinity purification. If theprotein of interest is not secreted by the cultured cells, then a lysisof the cultured cells should be performed prior to purification orisolation. One may use unclarified cell culture fluid containing theprotein of interest along with cell culture media components as well ascell culture additives, such as anti-foam compounds and other nutrientsand supplements, cells, cellular debris, host cell proteins, DNA,viruses and the like in the present invention. The process may beconducted in the bioreactor itself. The fluid may either bepreconditioned to a desired stimulus such as pH, temperature or otherstimulus characteristic or the fluid can be conditioned upon theaddition of polymer(s) or the polymer(s) can be added to a carrierliquid that is properly conditioned to the required parameter for thestimulus condition required for that polymer to be solubilized in thefluid. The polymer may be allowed to circulate thoroughly with the fluidand then the stimulus may be applied (change in pH, temperature, saltconcentration, etc) and the desired protein and polymer(s) precipitatecan out of the solution. The polymer and the desired protein(s) can beseparated from the rest of the fluid and optionally washed one or moretimes to remove any trapped or loosely bound contaminants. The desiredprotein may then be recovered from the polymer(s) by, for example,elution and the like. Preferably, the elution may be done under a set ofconditions such that the polymer remains in its precipitated form andretains any impurities to it during the selected elution of the desiredprotein. The polymer and protein as well as any impurities may besolubilized in a new fluid such as water or a buffered solution and theprotein may be recovered by a means such as affinity, ion exchanged,hydrophobic, or some other type of chromatography that has a preferenceand selectivity for the protein over that of the polymer or impurities.The eluted protein may then be recovered and may be subjected toadditional processing steps, either batch like steps or continuous flowthrough steps if appropriate.

In another embodiment, it may be useful to optimize the expression of aspecific polypeptide in a cell line or collection of cell lines ofpotential interest, particularly a polypeptide of interest such as aprotein variant of a reference protein having a known activity. In oneembodiment, provided is a method of optimizing expression of apolypeptide of interest in a target cell, by providing a plurality oftarget cell types, and independently contacting with each of theplurality of target cell types a modified mRNA encoding a polypeptide.Additionally, culture conditions may be altered to increase proteinproduction efficiency. Subsequently, the presence and/or level of thepolypeptide of interest in the plurality of target cell types isdetected and/or quantitated, allowing for the optimization of apolypeptide of interest's expression by selection of an efficient targetcell and cell culture conditions relating thereto. Such methods may beuseful when the polypeptide of interest contains one or morepost-translational modifications or has substantial tertiary structure,which often complicate efficient protein production.

Protein Recovery

The protein of interest may be preferably recovered from the culturemedium as a secreted polypeptide, or it can be recovered from host celllysates if expressed without a secretory signal. It may be necessary topurify the protein of interest from other recombinant proteins and hostcell proteins in a way that substantially homogenous preparations of theprotein of interest are obtained. The cells and/or particulate celldebris may be removed from the culture medium or lysate. The product ofinterest may then be purified from contaminant soluble proteins,polypeptides and nucleic acids by, for example, fractionation onimmunoaffinity or ion-exchange columns, ethanol precipitation, reversephase HPLC (RP-HPLC), SEPHADEX® chromatography, chromatography on silicaor on a cation exchange resin such as DEAE. Methods of purifying aprotein heterologous expressed by a host cell are well known in the art.

Methods and compositions described herein may be used to produceproteins which are capable of attenuating or blocking the endogenousagonist biological response and/or antagonizing a receptor or signalingmolecule in a mammalian subject. For example, IL-12 and IL-23 receptorsignaling may be enhanced in chronic autoimmune disorders such asmultiple sclerosis and inflammatory diseases such as rheumatoidarthritis, psoriasis, lupus erythematosus, ankylosing spondylitis andChron's disease (Kikly K, Liu L, Na S, Sedgwich J D (2006) Cur. Opin.Immunol. 18(6): 670-5). In another embodiment, a nucleic acid encodes anantagonist for chemokine receptors. Chemokine receptors CXCR-4 and CCR-5are required for HIV enry into host cells (Arenzana-Seisdedos F et al,(1996) Nature. October 3; 383 (6599):400).

VI. KITS Kits

The invention provides a variety of kits for conveniently and/oreffectively carrying out methods of the present invention. Typicallykits will comprise sufficient amounts and/or numbers of components toallow a user to perform multiple treatments of a subject(s) and/or toperform multiple experiments.

In one aspect, the present invention provides kits comprising themolecules (polynucleotides, primary constructs or mmRNA) of theinvention. In one embodiment, the kit comprises one or more functionalantibodies or function fragments thereof.

Said kits can be for protein production, comprising a firstpolynucleotide, primary construct or mmRNA comprising a translatableregion. The kit may further comprise packaging and instructions and/or adelivery agent to form a formulation composition. The delivery agent maycomprise a saline, a buffered solution, a lipidoid or any delivery agentdisclosed herein.

In one embodiment, the buffer solution may include sodium chloride,calcium chloride, phosphate and/or EDTA. In another embodiment, thebuffer solution may include, but is not limited to, saline, saline with2 mM calcium, 5% sucrose, 5% sucrose with 2 mM calcium, 5% Mannitol, 5%Mannitol with 2 mM calcium, Ringer's lactate, sodium chloride, sodiumchloride with 2 mM calcium. In a futher embodiment, the buffer solutionsmay be precipitated or it may be lyophilized. The amount of eachcomponent may be varied to enable consistent, reproducible higherconcentration saline or simple buffer formulations. The components mayalso be varied in order to increase the stability of modified RNA in thebuffer solution over a period of time and/or under a variety ofconditions.

In one aspect, the present invention provides kits for proteinproduction, comprising: a polynucleotide, primary construct or mmRNAcomprising a translatable region, provided in an amount effective toproduce a desired amount of a protein encoded by the translatable regionwhen introduced into a target cell; a second polynucleotide comprisingan inhibitory nucleic acid, provided in an amount effective tosubstantially inhibit the innate immune response of the cell; andpackaging and instructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a polynucleotide, primary construct or mmRNAcomprising a translatable region, wherein the polynucleotide exhibitsreduced degradation by a cellular nuclease, and packaging andinstructions.

In one aspect, the present invention provides kits for proteinproduction, comprising a polynucleotide, primary construct or mmRNAcomprising a translatable region, wherein the polynucleotide exhibitsreduced degradation by a cellular nuclease, and a mammalian cellsuitable for translation of the translatable region of the first nucleicacid.

In one aspect, the present invention provides kits for use in thesynthesis of chemically modified RNA, particularly mRNA which compriseone or more of the enzymes or enzyme variants identified using the PACEsystems.

VII. DEFINITIONS

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C₅ alkyl,and C6 alkyl.

About: As used herein, the term “about” means+/−10% of the recitedvalue.

Accessory plasmid: As used herein, the term “accessory plasmid” refersto a plasmid whose expression is dependent upon the activity of theevolving gene, in this case the evolving enzyme or enzyme variant. Onesuch plasmid is that of Liu and contains a rrnB 1 terminator, a promoterof interest, desired ribosome binding site, gene III, the bla gene andeither pUC or SC 101 origin of replication.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there may be an overlap of an effect of each agent on thepatient. In some embodiments, they are administered within about 60, 30,15, 10, 5, or 1 minute of one another. In some embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Antigens of interest or desired antigens: As used herein, the terms“antigens of interest” or “desired antigens” include those proteins andother biomolecules provided herein that are immunospecifically bound bythe antibodies and fragments, mutants, variants, and alterations thereofdescribed herein. Examples of antigens of interest include, but are notlimited to, insulin, insulin-like growth factor, hGH, tPA, cytokines,such as interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega orIFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF beta,TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Bifunctional: As used herein, the term “bifunctional” refers to anysubstance, molecule or moiety which is capable of or maintains at leasttwo functions. The functions may effect the same outcome or a differentoutcome. The structure that produces the function may be the same ordifferent. For example, bifunctional modified RNAs of the presentinvention may encode a cytotoxic peptide (a first function) while thosenucleosides which comprise the encoding RNA are, in and of themselves,cytotoxic (second function). In this example, delivery of thebifunctional modified RNA to a cancer cell would produce not only apeptide or protein molecule which may ameliorate or treat the cancer butwould also deliver a cytotoxic payload of nucleosides to the cell shoulddegradation, instead of translation of the modified RNA, occur.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, apolynucleotide, primary construct or mmRNA of the present invention maybe considered biologically active if even a portion of thepolynucleotide, primary construct or mmRNA is biologically active ormimics an activity considered biologically relevant.

Chemical terms: The following provides the definition of variouschemical terms from “acyl” to “thiol.”

The term “acyl,” as used herein, represents a hydrogen or an alkyl group(e.g., a haloalkyl group), as defined herein, that is attached to theparent molecular group through a carbonyl group, as defined herein, andis exemplified by formyl (i.e., a carboxyaldehyde group), acetyl,propionyl, butanoyl and the like. Exemplary unsubstituted acyl groupsinclude from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In someembodiments, the alkyl group is further substituted with 1, 2, 3, or 4substituents as described herein.

The term “acylamino,” as used herein, represents an acyl group, asdefined herein, attached to the parent molecular group though an aminogroup, as defined herein (i.e., —N(R^(N1))—C(O)—R, where R is H or anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group and R^(N1) isas defined herein). Exemplary unsubstituted acylamino groups includefrom 1 to 41 carbons (e.g., from 1 to 7, from 1 to 13, from 1 to 21,from 2 to 7, from 2 to 13, from 2 to 21, or from 2 to 41 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “acyloxy,” as used herein, represents an acyl group, as definedherein, attached to the parent molecular group though an oxygen atom(i.e., —O—C(O)—R, where R is H or an optionally substituted C₁₋₆, C₁₋₁₀,or C₁₋₂₀ alkyl group). Exemplary unsubstituted acyloxy groups includefrom 1 to 21 carbons (e.g., from 1 to 7 or from 1 to 11 carbons). Insome embodiments, the alkyl group is further substituted with 1, 2, 3,or 4 substituents as described herein, and/or the amino group is —NH₂ or—NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂,SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, or aryl, and each R^(N2) can beH, alkyl, or aryl.

The term “alkaryl,” as used herein, represents an aryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkaryl groups arefrom 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, suchas C₁₋₆ alk-C₆₋₁₀ aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀ alk-C₆₋₁₀ aryl).In some embodiments, the alkylene and the aryl each can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein forthe respective groups. Other groups preceded by the prefix “alk-” aredefined in the same manner, where “alk” refers to a C₁₋₆ alkylene,unless otherwise noted, and the attached chemical structure is asdefined herein.

The term “alkcycloalkyl” represents a cycloalkyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein (e.g., an alkylene group of from 1 to 4, from 1to 6, from 1 to 10, or form 1 to 20 carbons). In some embodiments, thealkylene and the cycloalkyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkenyl,” as used herein, represents monovalent straight orbranched chain groups of, unless otherwise specified, from 2 to 20carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one ormore carbon-carbon double bonds and is exemplified by ethenyl,1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, andthe like. Alkenyls include both cis and trans isomers. Alkenyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from amino, aryl, cycloalkyl, orheterocyclyl (e.g., heteroaryl), as defined herein, or any of theexemplary alkyl substituent groups described herein.

The term “alkenyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkenyl group (e.g., C₂₋₆ or C₂₋₁₀ alkenyl), unlessotherwise specified. Exemplary alkenyloxy groups include ethenyloxy,propenyloxy, and the like. In some embodiments, the alkenyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “alkheteroaryl” refers to a heteroaryl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheteroaryl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂heteroaryl, or C₁₋₂₀ alk-C₁₋₁₂ heteroaryl). In some embodiments, thealkylene and the heteroaryl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.Alkheteroaryl groups are a subset of alkheterocyclyl groups.

The term “alkheterocyclyl” represents a heterocyclyl group, as definedherein, attached to the parent molecular group through an alkylenegroup, as defined herein. Exemplary unsubstituted alkheterocyclyl groupsare from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heterocyclyl, C₁₋₁₀ alk-C₁₋₁₂heterocyclyl, or C₁₋₂₀ alk-C₁₋₁₂ heterocyclyl). In some embodiments, thealkylene and the heterocyclyl each can be further substituted with 1, 2,3, or 4 substituent groups as defined herein for the respective group.

The term “alkoxy” represents a chemical substituent of formula —OR,where R is a C₁₋₂₀ alkyl group (e.g., C₁₋₆ or C₁₋₁₀ alkyl), unlessotherwise specified. Exemplary alkoxy groups include methoxy, ethoxy,propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. Insome embodiments, the alkyl group can be further substituted with 1, 2,3, or 4 substituent groups as defined herein (e.g., hydroxy or alkoxy).

The term “alkoxyalkoxy” represents an alkoxy group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkoxy groupsinclude between 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20carbons, such as C₁₋₆ alkoxy-C₁₋₆ alkoxy, C₁₋₁₀ alkoxy-C₁₋₁₀ alkoxy, orC₁₋₂₀ alkoxy-C₁₋₂₀ alkoxy). In some embodiments, the each alkoxy groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkoxyalkyl” represents an alkyl group that is substitutedwith an alkoxy group. Exemplary unsubstituted alkoxyalkyl groups includebetween 2 to 40 carbons (e.g., from 2 to 12 or from 2 to 20 carbons,such as C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₁₀ alkoxy-C₁₋₁₀ alkyl, or C₁₋₂₀alkoxy-C₁₋₂₀ alkyl). In some embodiments, the alkyl and the alkoxy eachcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein for the respective group.

The term “alkoxycarbonyl,” as used herein, represents an alkoxy, asdefined herein, attached to the parent molecular group through acarbonyl atom (e.g., —C(O)—OR, where R is H or an optionally substitutedC₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplary unsubstitutedalkoxycarbonyl include from 1 to 21 carbons (e.g., from 1 to 11 or from1 to 7 carbons). In some embodiments, the alkoxy group is furthersubstituted with 1, 2, 3, or 4 substituents as described herein.

The term “alkoxycarbonylalkoxy,” as used herein, represents an alkoxygroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., —O-alkyl-C(O)—OR, where R is anoptionally substituted C₁₋₆, C₁₋₁₀, or C₁₋₂₀ alkyl group). Exemplaryunsubstituted alkoxycarbonylalkoxy include from 3 to 41 carbons (e.g.,from 3 to 10, from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31carbons, such as C₁₋₆ alkoxycarbonyl-C₁₋₆ alkoxy, C₁₋₁₀alkoxycarbonyl-C₁₋₁₀ alkoxy, or C₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkoxy). Insome embodiments, each alkoxy group is further independently substitutedwith 1, 2, 3, or 4 substituents, as described herein (e.g., a hydroxygroup).

The term “alkoxycarbonylalkyl,” as used herein, represents an alkylgroup, as defined herein, that is substituted with an alkoxycarbonylgroup, as defined herein (e.g., -alkyl-C(O)—OR, where R is an optionallysubstituted C₁₋₂₀, C₁₋₁₀, or C₁₋₆ alkyl group). Exemplary unsubstitutedalkoxycarbonylalkyl include from 3 to 41 carbons (e.g., from 3 to 10,from 3 to 13, from 3 to 17, from 3 to 21, or from 3 to 31 carbons, suchas C₁₋₆ alkoxycarbonyl-C₁₋₆ alkyl, C₁₋₁₀ alkoxycarbonyl-C₁₋₁₀ alkyl, orC₁₋₂₀ alkoxycarbonyl-C₁₋₂₀ alkyl). In some embodiments, each alkyl andalkoxy group is further independently substituted with 1, 2, 3, or 4substituents as described herein (e.g., a hydroxy group).

The term “alkyl,” as used herein, is inclusive of both straight chainand branched chain saturated groups from 1 to 20 carbons (e.g., from 1to 10 or from 1 to 6), unless otherwise specified. Alkyl groups areexemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- andtert-butyl, neopentyl, and the like, and may be optionally substitutedwith one, two, three, or, in the case of alkyl groups of two carbons ormore, four substituents independently selected from the group consistingof: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as definedherein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino(i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8)hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein. For example, the alkylene group of aC₁-alkaryl can be further substituted with an oxo group to afford therespective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent asaturated divalent hydrocarbon group derived from a straight or branchedchain saturated hydrocarbon by the removal of two hydrogen atoms, and isexemplified by methylene, ethylene, isopropylene, and the like. The term“C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylenegroups having between x and y carbons. Exemplary values for x are 1, 2,3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9,10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, orC₂₋₂₀ alkylene). In some embodiments, the alkylene can be furthersubstituted with 1, 2, 3, or 4 substituent groups as defined herein foran alkyl group.

The term “alkylsulfinyl,” as used herein, represents an alkyl groupattached to the parent molecular group through an —S(O)— group.Exemplary unsubstituted alkylsulfinyl groups are from 1 to 6, from 1 to10, or from 1 to 20 carbons. In some embodiments, the alkyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein.

The term “alkylsulfinylalkyl,” as used herein, represents an alkylgroup, as defined herein, substituted by an alkylsulfinyl group.Exemplary unsubstituted alkylsulfinylalkyl groups are from 2 to 12, from2 to 20, or from 2 to 40 carbons. In some embodiments, each alkyl groupcan be further substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “alkynyl,” as used herein, represents monovalent straight orbranched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bondand is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groupsmay be optionally substituted with 1, 2, 3, or 4 substituent groups thatare selected, independently, from aryl, cycloalkyl, or heterocyclyl(e.g., heteroaryl), as defined herein, or any of the exemplary alkylsubstituent groups described herein.

The term “alkynyloxy” represents a chemical substituent of formula —OR,where R is a C₂₋₂₀ alkynyl group (e.g., C₂₋₆ or C₂₋₁₀ alkynyl), unlessotherwise specified. Exemplary alkynyloxy groups include ethynyloxy,propynyloxy, and the like. In some embodiments, the alkynyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as definedherein (e.g., a hydroxy group).

The term “amidine,” as used herein, represents a —C(═NH)NH₂ group.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl,alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl, sulfoalkyl,heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g.,alkheteroaryl), wherein each of these recited R^(N1) groups can beoptionally substituted, as defined herein for each group; or two R^(N1)combine to form a heterocyclyl or an N-protecting group, and whereineach R^(N2) is, independently, H, alkyl, or aryl. The amino groups ofthe invention can be an unsubstituted amino (i.e., —NH₂) or asubstituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, aminois —NH₂ or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂,NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl,sulfoalkyl, or aryl, and each R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule havinga side chain, an amino group, and an acid group (e.g., a carboxy groupof —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attachedto the parent molecular group by the side chain, amino group, or acidgroup (e.g., the side chain). In some embodiments, the amino acid isattached to the parent molecular group by a carbonyl group, where theside chain or amino group is attached to the carbonyl group. Exemplaryside chains include an optionally substituted alkyl, aryl, heterocyclyl,alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl.Exemplary amino acids include alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine,hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline,ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine,taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groupsmay be optionally substituted with one, two, three, or, in the case ofamino acid groups of two carbons or more, four substituentsindependently selected from the group consisting of: (1) C₁₋₆ alkoxy;(2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g.,unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e.,—N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy;(9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′)is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d)hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g)polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, whereins1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), eachof s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is Hor C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15)—C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently,selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c)C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′)is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl,(c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), whereeach of R^(E′) and R^(F′) is, independently, selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from thegroup consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19)—NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consistingof (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from thegroup consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20)—NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the groupconsisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selectedfrom the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2)C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2)C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of—(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3,independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀alkyl, and (h2) amino-polyethylene glycol of—NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is aninteger from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 ands3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is,independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21)amidine. In some embodiments, each of these groups can be furthersubstituted as described herein.

The term “aminoalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aminoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by an amino group, as defined herein. Thealkyl and amino each can be further substituted with 1, 2, 3, or 4substituent groups as described herein for the respective group (e.g.,CO₂R^(A′), where R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl,e.g., carboxy).

The term “aryl,” as used herein, represents a mono-, bicyclic, ormulticyclic carbocyclic ring system having one or two aromatic rings andis exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl,1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl,indanyl, indenyl, and the like, and may be optionally substituted with1, 2, 3, 4, or 5 substituents independently selected from the groupconsisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl(e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl,halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy;(14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy);(17) —(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, andR^(A′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b)C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18)—(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four andwhere R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integer fromzero to four and where R^(D′) is selected from the group consisting of(a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and(27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can befurther substituted as described herein. For example, the alkylene groupof a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted withan oxo group to afford the respective aryloyl and (heterocyclyl)oylsubstituent group.

The term “arylalkoxy,” as used herein, represents an alkaryl group, asdefined herein, attached to the parent molecular group through an oxygenatom. Exemplary unsubstituted alkoxyalkyl groups include from 7 to 30carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₆₋₁₀aryl-C₁₋₆ alkoxy, C₆₋₁₀ aryl-C₁₋₁₀ alkoxy, or C₆₋₁₀ aryl-C₁₋₂₀ alkoxy).In some embodiments, the arylalkoxy group can be substituted with 1, 2,3, or 4 substituents as defined herein

The term “aryloxy” represents a chemical substituent of formula —OR′,where R′ is an aryl group of 6 to 18 carbons, unless otherwisespecified. In some embodiments, the aryl group can be substituted with1, 2, 3, or 4 substituents as defined herein.

The term “aryloyl,” as used herein, represents an aryl group, as definedherein, that is attached to the parent molecular group through acarbonyl group. Exemplary unsubstituted aryloyl groups are of 7 to 11carbons. In some embodiments, the aryl group can be substituted with 1,2, 3, or 4 substituents as defined herein.

The term “azido” represents an —N3 group, which can also be representedas —N═N═N.

The term “bicyclic,” as used herein, refer to a structure having tworings, which may be aromatic or non-aromatic. Bicyclic structuresinclude spirocyclyl groups, as defined herein, and two rings that shareone or more bridges, where such bridges can include one atom or a chainincluding two, three, or more atoms. Exemplary bicyclic groups include abicyclic carbocyclyl group, where the first and second rings arecarbocyclyl groups, as defined herein; a bicyclic aryl groups, where thefirst and second rings are aryl groups, as defined herein; bicyclicheterocyclyl groups, where the first ring is a heterocyclyl group andthe second ring is a carbocyclyl (e.g., aryl) or heterocyclyl (e.g.,heteroaryl) group; and bicyclic heteroaryl groups, where the first ringis a heteroaryl group and the second ring is a carbocyclyl (e.g., aryl)or heterocyclyl (e.g., heteroaryl) group. In some embodiments, thebicyclic group can be substituted with 1, 2, 3, or 4 substituents asdefined herein for cycloalkyl, heterocyclyl, and aryl groups.

The terms “carbocyclic” and “carbocyclyl,” as used herein, refer to anoptionally substituted C₃₋₁₂ monocyclic, bicyclic, or tricyclicstructure in which the rings, which may be aromatic or non-aromatic, areformed by carbon atoms. Carbocyclic structures include cycloalkyl,cycloalkenyl, and aryl groups.

The term “carbamoyl,” as used herein, represents —C(O)—N(R^(N1))₂, wherethe meaning of each R^(N1) is found in the definition of “amino”provided herein.

The term “carbamoylalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carbamoyl group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “carbamyl,” as used herein, refers to a carbamate group havingthe structure —NR^(N1)C(═O)OR or —OC(═O)N(R^(N1))₂, where the meaning ofeach R^(N1) is found in the definition of “amino” provided herein, and Ris alkyl, cycloalkyl, alkcycloalkyl, aryl, alkaryl, heterocyclyl (e.g.,heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), as definedherein.

The term “carbonyl,” as used herein, represents a C(O) group, which canalso be represented as C═O.

The term “carboxyaldehyde” represents an acyl group having the structure—CHO.

The term “carboxy,” as used herein, means —CO₂H.

The term “carboxyalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkoxy group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein for the alkyl group.

The term “carboxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a carboxy group, as defined herein. Thealkyl group can be further substituted with 1, 2, 3, or 4 substituentgroups as described herein.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkoxy” represents a chemical substituent of formula —OR,where R is a C₃₋₈ cycloalkyl group, as defined herein, unless otherwisespecified. The cycloalkyl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein. Exemplary unsubstitutedcycloalkoxy groups are from 3 to 8 carbons. In some embodiment, thecycloalkyl group can be further substituted with 1, 2, 3, or 4substituent groups as described herein.

The term “cycloalkyl,” as used herein represents a monovalent saturatedor unsaturated non-aromatic cyclic hydrocarbon group from three to eightcarbons, unless otherwise specified, and is exemplified by cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1.]heptyl,and the like. When the cycloalkyl group includes one carbon-carbondouble bond, the cycloalkyl group can be referred to as a “cycloalkenyl”group. Exemplary cycloalkenyl groups include cyclopentenyl,cyclohexenyl, and the like. The cycloalkyl groups of this invention canbe optionally substituted with: (1) C₁₋₇ acyl (e.g., carboxyaldehyde);(2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl,(carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g., perfluoroalkyl),hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl);(3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such as perfluoroalkoxy); (4) C₁₋₆alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8)azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo;(12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g.,C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q is an integer fromzero to four, and R^(A′) is selected from the group consisting of (a)C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl;(18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to fourand where R^(B′) and R^(C′) are independently selected from the groupconsisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d)C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integerfrom zero to four and where R^(D′) is selected from the group consistingof (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20)—(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four andwhere each of R^(E′) and R^(F′) is, independently, selected from thegroup consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and(d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of thesegroups can be further substituted as described herein. For example, thealkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be furthersubstituted with an oxo group to afford the respective aryloyl and(heterocyclyl)oyl substituent group.

The term “diastereomer,” as used herein means stereoisomers that are notmirror images of one another and are non-superimposable on one another.

The term “effective amount” of an agent, as used herein, is that amountsufficient to effect beneficial or desired results, for example,clinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. For example, in the context ofadministering an agent that treats cancer, an effective amount of anagent is, for example, an amount sufficient to achieve treatment, asdefined herein, of cancer, as compared to the response obtained withoutadministration of the agent.

The term “enantiomer,” as used herein, means each individual opticallyactive form of a compound of the invention, having an optical purity orenantiomeric excess (as determined by methods standard in the art) of atleast 80% (i.e., at least 90% of one enantiomer and at most 10% of theother enantiomer), preferably at least 90% and more preferably at least98%.

The term “halo,” as used herein, represents a halogen selected frombromine, chlorine, iodine, or fluorine.

The term “haloalkoxy,” as used herein, represents an alkoxy group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkoxy may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkoxy groupsinclude perfluoroalkoxys (e.g., —OCF₃), —OCHF₂, —OCH₂F, —OCCl₃,—OCH₂CH₂Br, —OCH₂CH(CH₂CH₂Br)CH₃, and —OCHICH₃. In some embodiments, thehaloalkoxy group can be further substituted with 1, 2, 3, or 4substituent groups as described herein for alkyl groups.

The term “haloalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkyl may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkyl groupsinclude perfluoroalkyls (e.g., —CF₃), —CHF₂, —CH₂F, —CCl₃, —CH₂CH₂Br,—CH₂CH(CH₂CH₂Br)CH₃, and —CHICH₃. In some embodiments, the haloalkylgroup can be further substituted with 1, 2, 3, or 4 substituent groupsas described herein for alkyl groups.

The term “heteroalkylene,” as used herein, refers to an alkylene group,as defined herein, in which one or two of the constituent carbon atomshave each been replaced by nitrogen, oxygen, or sulfur. In someembodiments, the heteroalkylene group can be further substituted with 1,2, 3, or 4 substituent groups as described herein for alkylene groups.

The term “heteroaryl,” as used herein, represents that subset ofheterocyclyls, as defined herein, which are aromatic: i.e., they contain4n+2 pi electrons within the mono- or multicyclic ring system. Exemplaryunsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10,1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In someembodiment, the heteroaryl is substituted with 1, 2, 3, or 4substituents groups as defined for a heterocyclyl group.

The term “heterocyclyl,” as used herein represents a 5-, 6- or7-membered ring, unless otherwise specified, containing one, two, three,or four heteroatoms independently selected from the group consisting ofnitrogen, oxygen, and sulfur. The 5-membered ring has zero to two doublebonds, and the 6- and 7-membered rings have zero to three double bonds.Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. Theterm “heterocyclyl” also represents a heterocyclic compound having abridged multicyclic structure in which one or more carbons and/orheteroatoms bridges two non-adjacent members of a monocyclic ring, e.g.,a quinuclidinyl group. The term “heterocyclyl” includes bicyclic,tricyclic, and tetracyclic groups in which any of the above heterocyclicrings is fused to one, two, or three carbocyclic rings, e.g., an arylring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, acyclopentene ring, or another monocyclic heterocyclic ring, such asindolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl,benzothienyl and the like. Examples of fused heterocyclyls includetropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics includepyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl,pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl,morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl,isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl,quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl,benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl,triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl,thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl,dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl,dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl,isobenzofuranyl, benzothienyl, and the like, including dihydro andtetrahydro forms thereof, where one or more double bonds are reduced andreplaced with hydrogens. Still other exemplary heterocyclyls include:2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl;2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g.,2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl);2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g.,2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl);2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g.,2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl);4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl);2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl);1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g.,2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl);1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g.,1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl);1,6-dihydro-6-oxo-pyridazinyl (e.g.,1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl(e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl);2,3-dihydro-2-oxo-1H-indolyl (e.g.,3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl);1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl;1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl);2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g.,3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl);2,3-dihydro-2-oxo-benzoxazolyl (e.g.,5-chloro-2,3-dihydro-2-oxo-benzoxazolyl);2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl;1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g.,2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl);1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g.,1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl);1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g.,1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl);1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g.,1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl);2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and1,8-naphthylenedicarboxamido. Additional heterocyclics include3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl),tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl,thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups alsoinclude groups of the formula

where

E′ is selected from the group consisting of —N— and —CH—; F′ is selectedfrom the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—, —CH═N—,—CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—, —O—, and—S—; and G′ is selected from the group consisting of —CH— and —N—. Anyof the heterocyclyl groups mentioned herein may be optionallysubstituted with one, two, three, four or five substituentsindependently selected from the group consisting of: (1) C₁₋₇ acyl(e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl,azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g.,perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such asperfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7)C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂₋₁₂ heteroaryl);(13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q isan integer from zero to four, and R^(A′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integerfrom zero to four and where R^(B′) and R^(C′) are independently selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q isan integer from zero to four and where R^(D′) is selected from the groupconsisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zeroto four and where each of R^(E′) and R^(F′) is, independently, selectedfrom the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23)C₃₋₈ cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl(e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In someembodiments, each of these groups can be further substituted asdescribed herein. For example, the alkylene group of a C₁-alkaryl or aC₁-alkheterocyclyl can be further substituted with an oxo group toafford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “(heterocyclyl)imino,” as used herein, represents aheterocyclyl group, as defined herein, attached to the parent moleculargroup through an imino group. In some embodiments, the heterocyclylgroup can be substituted with 1, 2, 3, or 4 substituent groups asdefined herein.

The term “(heterocyclyl)oxy,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group throughan oxygen atom. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “(heterocyclyl)oyl,” as used herein, represents a heterocyclylgroup, as defined herein, attached to the parent molecular group througha carbonyl group. In some embodiments, the heterocyclyl group can besubstituted with 1, 2, 3, or 4 substituent groups as defined herein.

The term “hydrocarbon,” as used herein, represents a group consistingonly of carbon and hydrogen atoms.

The term “hydroxy,” as used herein, represents an —OH group.

The term “hydroxyalkenyl,” as used herein, represents an alkenyl group,as defined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by dihydroxypropenyl,hydroxyisopentenyl, and the like.

The term “hydroxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by hydroxymethyl,dihydroxypropyl, and the like.

The term “isomer,” as used herein, means any tautomer, stereoisomer,enantiomer, or diastereomer of any compound of the invention. It isrecognized that the compounds of the invention can have one or morechiral centers and/or double bonds and, therefore, exist asstereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers)or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/transisomers). According to the invention, the chemical structures depictedherein, and therefore the compounds of the invention, encompass all ofthe corresponding stereoisomers, that is, both the stereomerically pureform (e.g., geometrically pure, enantiomerically pure, ordiastereomerically pure) and enantiomeric and stereoisomeric mixtures,e.g., racemates. Enantiomeric and stereoisomeric mixtures of compoundsof the invention can typically be resolved into their componentenantiomers or stereoisomers by well-known methods, such as chiral-phasegas chromatography, chiral-phase high performance liquid chromatography,crystallizing the compound as a chiral salt complex, or crystallizingthe compound in a chiral solvent. Enantiomers and stereoisomers can alsobe obtained from stereomerically or enantiomerically pure intermediates,reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group,as defined herein, to which is attached one or two N-protecting groups,as defined herein.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups, such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

The term “nitro,” as used herein, represents an —NO₂ group.

The term “oxo” as used herein, represents ═O.

The term “perfluoroalkyl,” as used herein, represents an alkyl group, asdefined herein, where each hydrogen radical bound to the alkyl group hasbeen replaced by a fluoride radical. Perfluoroalkyl groups areexemplified by trifluoromethyl, pentafluoroethyl, and the like.

The term “perfluoroalkoxy,” as used herein, represents an alkoxy group,as defined herein, where each hydrogen radical bound to the alkoxy grouphas been replaced by a fluoride radical. Perfluoroalkoxy groups areexemplified by trifluoromethoxy, pentafluoroethoxy, and the like.

The term “spirocyclyl,” as used herein, represents a C₂₋₇ alkylenediradical, both ends of which are bonded to the same carbon atom of theparent group to form a spirocyclic group, and also a C₁₋₆ heteroalkylenediradical, both ends of which are bonded to the same atom. Theheteroalkylene radical forming the spirocyclyl group can containing one,two, three, or four heteroatoms independently selected from the groupconsisting of nitrogen, oxygen, and sulfur. In some embodiments, thespirocyclyl group includes one to seven carbons, excluding the carbonatom to which the diradical is attached. The spirocyclyl groups of theinvention may be optionally substituted with 1, 2, 3, or 4 substituentsprovided herein as optional substituents for cycloalkyl and/orheterocyclyl groups.

The term “stereoisomer,” as used herein, refers to all possibledifferent isomeric as well as conformational forms which a compound maypossess (e.g., a compound of any formula described herein), inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

The term “sulfoalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a sulfo group of —SO₃H. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thioalkaryl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkaryl group. In someembodiments, the alkaryl group can be further substituted with 1, 2, 3,or 4 substituent groups as described herein.

The term “thioalkheterocyclyl,” as used herein, represents a chemicalsubstituent of formula —SR, where R is an alkheterocyclyl group. In someembodiments, the alkheterocyclyl group can be further substituted with1, 2, 3, or 4 substituent groups as described herein.

The term “thioalkoxy,” as used herein, represents a chemical substituentof formula —SR, where R is an alkyl group, as defined herein. In someembodiments, the alkyl group can be further substituted with 1, 2, 3, or4 substituent groups as described herein.

The term “thiol” represents an —SH group.

Chimeric Enzyme: As used herein, the term “chimeric enzyme” or “fusionenzyme” refers to an enzyme that is not a native enzyme found in nature.

Compound: As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone-enol pairs, amide-imidicacid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an oligonucleotide orpolypeptide or may apply to a portion, region or feature thereof.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the engineered RNA or mRNA of the present invention may be singleunits or multimers or comprise one or more components of a complex orhigher order structure.

Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing,suppressing the growth, division, or multiplication of a cell (e.g., amammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of apolynucleotide, primary construct or mmRNA to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Dose splitting factor (DSF)-ratio of PUD of dose split treatment dividedby PUD of total daily dose or single unit dose. The value is derivedfrom comparison of dosing regimens groups.

Encoded protein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence which encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point, wildtype or native molecule.

Evolution: As used herein, the term “evolution” refers to process ofchange that results in the production of nucleic acids and polypeptidesthat retain at least some of the structural features or elements and/orfunctional activity of the parent nucleic acid or polypeptide from whichthey have developed.

Exosome: As used herein, “exosome” is a vesicle secreted by mammaliancells or a complex involved in RNA degradation.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least apolynucleotide, primary construct or mmRNA and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the invention, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the invention, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between oligonucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof. Methods forisolating compounds and their salts are routine in the art.

Linker: As used herein, a linker refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to a modifiednucleoside or nucleotide on the nucleobase or sugar moiety at a firstend, and to a payload, e.g., a detectable or therapeutic agent, at asecond end. The linker may be of sufficient length as to not interferewith incorporation into a nucleic acid sequence. The linker can be usedfor any useful purpose, such as to form mmRNA multimers (e.g., throughlinkage of two or more polynucleotides, primary constructs, or mmRNAmolecules) or mmRNA conjugates, as well as to administer a payload, asdescribed herein. Examples of chemical groups that can be incorporatedinto the linker include, but are not limited to, alkyl, alkenyl,alkynyl, amido, amino, ether, thioether, ester, alkylene,heteroalkylene, aryl, or heterocyclyl, each of which can be optionallysubstituted, as described herein. Examples of linkers include, but arenot limited to, unsaturated alkanes, polyethylene glycols (e.g.,ethylene or propylene glycol monomeric units, e.g., diethylene glycol,dipropylene glycol, triethylene glycol, tripropylene glycol,tetraethylene glycol, or tetraethylene glycol), and dextran polymers,Other examples include, but are not limited to, cleavable moietieswithin the linker, such as, for example, a disulfide bond (—S—S—) or anazo bond (—N═N—), which can be cleaved using a reducing agent orphotolysis. Non-limiting examples of a selectively cleavable bondinclude an amido bond can be cleaved for example by the use oftris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/orphotolysis, as well as an ester bond can be cleaved for example byacidic or basic hydrolysis.

Methylation: As used herein when referring to modifications,“methylation” refers to the addition of a methyl group.

MicroRNA (miRNA) binding site: As used herein, a microRNA (miRNA)binding site represents a nucleotide location or region of a nucleicacid transcript to which at least the “seed” region of a miRNA binds.

Modified: As used herein “modified” refers to a changed state orstructure of a molecule of the invention. Molecules may be modified inmany ways including chemically, structurally, and functionally. In oneembodiment, the mRNA molecules of the present invention are modified bythe introduction of non-natural nucleosides and/or nucleotides, e.g., asit relates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “modified”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Mucus: As used herein, “mucus” refers to the natural substance that isviscous and comprises mucin glycoproteins.

Mutagenesis plasmid: As used herein, “mutagenesis plasmid” refers to aplasmid which may cause mutations in a host cell.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Non-human vertebrate: As used herein, a “non human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g. alkyl) per se is optional.

Paratope: As used herein, a “paratope” refers to the antigen-bindingsite of an antibody.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Phage-assisted continuous evolution: As used herein, the phrase“phage-assisted continuous evolution” referes to the process of evolvingnucleic acids or polypeptides by transferring nucleic acids orpolypeptides from host cell to host cell through a modifiedbacteriophage life cycle.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is modified byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17^(th) ed., Mack Publishing Company, Easton,Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, andUse, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge etal., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of whichis incorporated herein by reference in its entirety.

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestered in some wayand which release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Pseudouridylation: As used herein when referring to modifications,“pseudouridylation” is achieved by the isomerization of uridine usingpseudouridine synthetase.

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

RNA Polymerase: As used herein, the term “RNA polymerase” refers to anenzyme that is capable of synthesizing RNA from a DNA template.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Selection Plasmid: As used herein, the term “selection plasmid” refersto a plasmid used in the PACE system which encodes the variant libraryof potential new enzymes against which selection will be made. One suchplasmid example is that of Liu comprising replacing all but the last 180base pairs of gene II with the gene encoding T7 RNA polymerase in aVSCM13 helper phage.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence which can direct the transport or localization of aprotein.

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administered in one dose/at one time/single route/singlepoint of contact, i.e., single administration event.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Unmodified: As used herein, “unmodified” refers to any substance,compound or molecule prior to being changed in any way. Unmodified may,but does not always, refer to the wild type or native form of abiomolecule. Molecules may undergo a series of modifications wherebyeach modified molecule may serve as the “unmodified” starting moleculefor a subsequent modification.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

All cited sources, for example, references, publications, databases,database entries, and art cited herein, are incorporated into thisapplication by reference, even if not expressly stated in the citation.In case of conflicting statements of a cited source and the instantapplication, the statement in the instant application shall control.

Section and table headings are not intended to be limiting.

EXAMPLES Example 1 Modified mRNA Production

Modified mRNAs (mmRNA) according to the invention may be made usingstandard laboratory methods and materials. The open reading frame (ORF)of the gene of interest may be flanked by a 5′ untranslated region (UTR)which may contain a strong Kozak translational initiation signal and/oran alpha-globin 3′ UTR which may include an oligo(dT) sequence fortemplated addition of a poly-A tail. The modified mRNAs may be modifiedto reduce the cellular innate immune response.

The ORF may also include various upstream or downstream additions (suchas, but not limited to, β-globin, tags, etc.) may be ordered from anoptimization service such as, but limited to, DNA2.0 (Menlo Park,Calif.) and may contain multiple cloning sites which may have XbaIrecognition. Upon receipt of the construct, it may be reconstituted andtransformed into chemically competent E. coli.

For the present invention, NEB DH5-alpha Competent E. coli may be used.Transformations are performed according to NEB instructions using 100 ngof plasmid. The protocol is as follows:

-   -   1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for        10 minutes.    -   2. Add 1-5 μA containing 1 pg-100 ng of plasmid DNA to the cell        mixture. Carefully flick the tube 4-5 times to mix cells and        DNA. Do not vortex.    -   3. Place the mixture on ice for 30 minutes. Do not mix.    -   4. Heat shock at 42° C. for exactly 30 seconds. Do not mix.    -   5. Place on ice for 5 minutes. Do not mix.    -   6. Pipette 950 μl of room temperature SOC into the mixture.    -   7. Place at 37° C. for 60 minutes. Shake vigorously (250 rpm) or        rotate.    -   8. Warm selection plates to 37° C.    -   9. Mix the cells thoroughly by flicking the tube and inverting.    -   10. Spread 50-100 μL of each dilution onto a selection plate and        incubate overnight at 37° C. Alternatively, incubate at 30° C.        for 24-36 hours or 25° C. for 48 hours.

A single colony is then used to inoculate 5 ml of LB growth media usingthe appropriate antibiotic and then allowed to grow (250 RPM, 37° C.)for 5 hours. This is then used to inoculate a 200 ml culture medium andallowed to grow overnight under the same conditions.

To isolate the plasmid (up to 850 μg), a maxi prep is performed usingthe Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.),following the manufacturer's instructions.

In order to generate cDNA for In Vitro Transcription (IVT), the plasmid(an Example of which is shown in FIG. 3) is first linearized using arestriction enzyme such as XbaI. A typical restriction digest with XbaIwill comprise the following: Plasmid 1.0 μg; 10× Buffer 1.0 μL; XbaI 1.5μL; dH₂O up to 10 μl; incubated at 37° C. for 1 hr. If performing at labscale (<5 μg), the reaction is cleaned up using Invitrogen's PURELINK™PCR Micro Kit (Carlsbad, Calif.) per manufacturer's instructions. Largerscale purifications may need to be done with a product that has a largerload capacity such as Invitrogen's standard PURELINK™ PCR Kit (Carlsbad,Calif.). Following the cleanup, the linearized vector is quantifiedusing the NanoDrop and analyzed to confirm linearization using agarosegel electrophoresis.

As a non-limiting example, G-CSF may represent the polypeptide ofinterest. Representative sequences which may be used in the stepsoutlined in Examples 1-5 are shown in Table 11. It should be noted thatthe start codon (ATG) has been underlined in each sequence of Table 11.

TABLE 11 G-CSF Sequences SEQ ID NO Description 168 cDNAsequence:ATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCC CTGA 169 cDNA having T7polymerase site, AfeI and Xba restriction site:TAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCTGGACCTGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGTGCACTCTGGACAGTGCAGGAAGCCACCCCCCTGGGCCCTGCCAGCTCCCTGCCCCAGAGCTTCCTGCTCAAGTGCTTAGAGCAAGTGAGGAAGATCCAGGGCGATGGCGCAGCGCTCCAGGAGAAGCTGTGTGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTCGGACACTCTCTGGGCATCCCCTGGGCTCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCAGGCTGCTTGAGCCAACTCCATAGCGGCCTTTTCCTCTACCAGGGGCTCCTGCAGGCCCTGGAAGGGATCTCCCCCGAGTTGGGTCCCACCTTGGACACACTGCAGCTGGACGTCGCCGACTTTGCCACCACCATCTGGCAGCAGATGGAAGAACTGGGAATGGCCCCTGCCCTGCAGCCCACCCAGGGTGCCATGCCGGCCTTCGCCTCTGCTTTCCAGCGCCGGGCAGGAGGGGTCCTGGTTGCCTCCCATCTGCAGAGCTTCCTGGAGGTGTCGTACCGCGTTCTACGCCACCTTGCCCAGCCCTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCT AGA 170 Optimizedsequence; containing T7 polymerase site, AfeI and Xba restriction siteTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGGTCCCGCGACCCAAAGCCCCATGAAACTTATGGCCCTGCAGTTGCTGCTTTGGCACTCGGCCCTCTGGACAGTCCAAGAAGCGACTCCTCTCGGACCTGCCTCATCGTTGCCGCAGTCATTCCTTTTGAAGTGTCTGGAGCAGGTGCGAAAGATTCAGGGCGATGGAGCCGCACTCCAAGAGAAGCTCTGCGCGACATACAAACTTTGCCATCCCGAGGAGCTCGTACTGCTCGGGCACAGCTTGGGGATTCCCTGGGCTCCTCTCTCGTCCTGTCCGTCGCAGGCTTTGCAGTTGGCAGGGTGCCTTTCCCAGCTCCACTCCGGTTTGTTCTTGTATCAGGGACTGCTGCAAGCCCTTGAGGGAATCTCGCCAGAATTGGGCCCGACGCTGGACACGTTGCAGCTCGACGTGGCGGATTTCGCAACAACCATCTGGCAGCAGATGGAGGAACTGGGGATGGCACCCGCGCTGCAGCCCACGCAGGGGGCAATGCCGGCCTTTGCGTCCGCGTTTCAGCGCAGGGCGGGTGGAGTCCTCGTAGCGAGCCACCTTCAATCATTTTTGGAAGTCTCGTACCGGGTGCTGAGACATCTTGCGCAGCCGTGAAGCGCTGCCTTCTGCGGGGCTTGCCTTCTGGCCATGCCCTTCTTCTCTCCCTTGCACCTGTACCTCTTGGTCTTTGAATAAAGCCTGAGTAGGAAGGCGGCCGCTCGAGCATGCATCT AGA 171 mRNA sequence(transcribed) GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGCCGGUCCCGCGACCCAAAGCCCCAUGAAACUUAUGGCCCUGCAGUUGCUGCUUUGGCACUCGGCCCUCUGGACAGUCCAAGAAGCGACUCCUCUCGGACCUGCCUCAUCGUUGCCGCAGUCAUUCCUUUUGAAGUGUCUGGAGCAGGUGCGAAAGAUUCAGGGCGAUGGAGCCGCACUCCAAGAGAAGCUCUGCGCGACAUACAAACUUUGCCAUCCCGAGGAGCUCGUACUGCUCGGGCACAGCUUGGGGAUUCCCUGGGCUCCUCUCUCGUCCUGUCCGUCGCAGGCUUUGCAGUUGGCAGGGUGCCUUUCCCAGCUCCACUCCGGUUUGUUCUUGUAUCAGGGACUGCUGCAAGCCCUUGAGGGAAUCUCGCCAGAAUUGGGCCCGACGCUGGACACGUUGCAGCUCGACGUGGCGGAUUUCGCAACAACCAUCUGGCAGCAGAUGGAGGAACUGGGGAUGGCACCCGCGCUGCAGCCCACGCAGGGGGCAAUGCCGGCCUUUGCGUCCGCGUUUCAGCGCAGGGCGGGUGGAGUCCUCGUAGCGAGCCACCUUCAAUCAUUUUUGGAAGUCUCGUACCGGGUGCUGAGACAUCUUGCGCAGCC GUGAAGCGCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGA AG

Example 2 PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix12.5 μl; Forward Primer (10 uM) 0.75 μl; ReversePrimer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH₂O diluted to 25.0μl. The reaction conditions are at 95° C. for 5 min. and 25 cycles of98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45 sec, then72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention incorporates a poly-T₁₂₀ fora poly-Aim in the mRNA. Other reverse primers with longer or shorterpoly(T) tracts can be used to adjust the length of the poly(A) tail inthe mRNA.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 3 In vitro Transcription (IVT)

The in vitro transcription reaction generates mRNA containing modifiednucleotides or modified RNA. The input nucleotide triphosphate (NTP) mixis made in-house using natural and un-natural NTPs. The IVT reaction mayemploy one or more of the novel polymerase enzyme variants identifiedusing the PACE protocol.

A typical in vitro transcription reaction includes the following:

1. Template cDNA 1.0 μg 2. 10x transcription buffer (400 mM Tris-HCl 2.0μl pH 8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 3. Custom NTPs (25mM each) 7.2 μl 4. RNase Inhibitor 20 U 5. T7 RNA polymerase (wild typeor variant) 3000 U 6. dH₂0 Up to 20.0 μl. and 7. Incubation at 37° C.for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 4 Enzymatic Capping of mRNA

Capping of the mRNA is performed as follows where the mixture includes:IVT RNA 60 μg-180 μg and dH₂0 up to 72 μl. The mixture is incubated at65° C. for 5 minutes to denature RNA, and then is transferredimmediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (wild type or enzyme variant) (400U); Vacciniacapping enzyme (Guanylyl transferase) (40 U); dH₂0 (Up to 28 μl); andincubation at 37° C. for 30 minutes for 60 μg RNA or up to 2 hours for180 μg of RNA.

The mRNA is then purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.)following the manufacturer's instructions. Following the cleanup, theRNA is quantified using the NANODROP™ (ThermoFisher, Waltham, Mass.) andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred. The RNA productmay also be sequenced by running a reverse-transcription-PCR to generatethe cDNA for sequencing.

Example 5 PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (wild type or PACE enzyme variant) (20 U); dH₂0up to 123.5 μl and incubation at 37° C. for 30 min. If the poly-A tailis already in the transcript, then the tailing reaction may be skippedand proceed directly to cleanup with Ambion's MEGACLEAR™ kit (Austin,Tex.) (up to 500 μg). Poly-A Polymerase is preferably a recombinantenzyme expressed in yeast.

For studies performed and described herein, the poly-A tail is encodedin the IVT template to comprise 160 nucleotides in length. However, itshould be understood that the processivity or integrity of the polyAtailing reaction may not always result in exactly 160 nucleotides. HencepolyA tails of approximately 160 nucleotides, e.g, about 150-165, 155,156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scopeof the invention.

Example 6 Natural 5′ Caps and 5′ Cap Analogues

5′-capping of modified RNA may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzyme(wild type or PACE enzyme variant) to generate the “Cap 0” structure:m7G(5′)ppp(5′)G (New England BioLabs, Ipswich, Mass.). Cap 1 structuremay be generated using both Vaccinia Virus Capping Enzyme and a 2′-Omethyl-transferase (wild type or PACE enzyme variant) to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 7 Capping

A. Protein Expression Assay

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 168; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 171with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA (3′ O-Me-m7G(5)ppp(5′)G) cap analog or theCap1 structure can be transfected into human primary keratinocytes atequal concentrations. 6, 12, 24 and 36 hours post-transfection theamount of G-CSF secreted into the culture medium can be assayed byELISA. Synthetic mRNAs that secrete higher levels of G-CSF into themedium would correspond to a synthetic mRNA with a highertranslationally-competent Cap structure.

B. Purity Analysis Synthesis

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 168; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 171with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA cap analog or the Cap1 structure crudesynthesis products can be compared for purity using denaturingAgarose-Urea gel electrophoresis or HPLC analysis. Synthetic mRNAs witha single, consolidated band by electrophoresis correspond to the higherpurity product compared to a synthetic mRNA with multiple bands orstreaking bands. Synthetic mRNAs with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure mRNA population.

C. Cytokine Analysis

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 168; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 171with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can betransfected into human primary keratinocytes at multiple concentrations.6, 12, 24 and 36 hours post-transfection the amount of pro-inflammatorycytokines such as TNF-alpha and IFN-beta secreted into the culturemedium can be assayed by ELISA. Synthetic mRNAs that secrete higherlevels of pro-inflammatory cytokines into the medium would correspond toa synthetic mRNA containing an immune-activating cap structure.

D. Capping Reaction Efficiency

Synthetic mRNAs encoding human G-CSF (cDNA shown in SEQ ID NO: 168; mRNAsequence fully modified with 5-methylcytosine at each cytosine andpseudouridine replacement at each uridine site shown in SEQ ID NO: 171with a polyA tail approximately 160 nucleotides in length not shown insequence) containing the ARCA cap analog or the Cap1 structure can beanalyzed for capping reaction efficiency by LC-MS after capped mRNAnuclease treatment. Nuclease treatment of capped mRNAs would yield amixture of free nucleotides and the capped 5′-5-triphosphate capstructure detectable by LC-MS. The amount of capped product on the LC-MSspectra can be expressed as a percent of total mRNA from the reactionand would correspond to capping reaction efficiency. The cap structurewith higher capping reaction efficiency would have a higher amount ofcapped product by LC-MS.

Example 8 Agarose Gel Electrophoresis of Modified RNA or RT PCR Products

Individual modified RNAs (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) are loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes according to the manufacturer protocol.

Example 9 Nanodrop Modified RNA Quantification and UV Spectral Data

Modified RNAs in TE buffer (1 μl) are used for Nanodrop UV absorbancereadings to quantitate the yield of each modified RNA from an in vitrotranscription reaction.

Example 10 Phage Assisted Continuous Evolution

Phage-assisted continuous evolution (PACE) may be used to evolve enzymesfor the production of and use of modified mRNA in in vitro transcriptionand post transcriptional reactoins. Any number of enzymes of the IVTreactions and post-IVT reactions may be replaced by novel enzyme mutantsor variants. These include RNA polymerases, capping enzymes,transferases (including 2′O-methyl transferases and post transcriptionaltransferases), synthetases and the like.

Accordingly, novel enzymes or enzyme mutants are produced whichfacilitate improved production and/or manufacture of chemically modifiedmRNA using the methods described by Esvelt et al (Nature, 2011472(7344); 499-503) and in U.S. Patent Application No. 20110177495, thecontents of which are incorporated herein by reference in theirentirety. The PACE process is briefly described below (A-C). It isunderstood that certain modifications may be made to the buffers andreagents, especially in the use of chemically modified NTPs necessary toevolve RNA polymerases which are permissive to the modified NTPs.

A. Phage Assisted Continuous Evolution

The E. coli host cells containing an accessory plasmid and mutagenesisplasmid (inflow), one that induces mutagenesis in the host cell, arepumped at a fixed volume into a fixed-volume lagoon which is seeded witha selection phage. The accessory plasmid comprises the T7 promoter andthe gene III protein. In the selection phage all but the last 180 basepairs of the gene III protein are replaced with the gene encoding the T7RNA polymerase. Aliquots of the lagoon and the inflow are taken regularto monitor the evolution of the T7 RNA polymerase. The evolved mutantsare isolated and subcloned for further studies in cell-based or in vitroactivity assays.

B. Cell-Based Activity Assays

Overnight cultures of cells grown in 2×YT media are diluted 4-fold infresh 2×YT media. 20 uL of the diluted culture is mixed with 80 uL ofbuffer at a pH of 7.0 comprising 60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mMKCl, 1 mM MgSO4 and 50 mM of beta-mercaptoethanol in assay plates. Theabsorbance at 595 nm is measured using a plate reader. 25 uL of 1 mg/mLmethylumbelliferyl-beta-(D)-galactopyranoside is added to each well.Plates are incubated at 30° C. and fluorescence is measure at 360/460 nmon a plate reader. Plates are measured at multiple time points to avoidsaturation of the spectrophotometer or consumption of the substrate.

C. In Vitro Activity Assays

Purified T7 RNA polymerase variant concentration are determined byBradford assay and then by Coomassie stain on a 4-12% NuPage gel.Templates are prepared by PCR amplification of 150 bp fragments of thereporter plasmid used for in vivo assays. Templates are purified bymethods known in the art and transcription reactions are performed in1×RNA polymerase buffer comprising 40 mM Tris-HCl, 6 mM MgCl₂, 10 mMdithiothreitol, 2 mM spermidine at a pH of 7.9 with 1 mM ofribonucleotide triphosphate (rNTP), 1 ng template DNA, purifiedpolymerase variant, and 2mCi[α-³²P]ATP. Reactions are incubated at 37°C. for 20 minutes, mixed with an equivalent volume of loading dyecomprising 7M urea, 178 mM Tris-C1, 178 mMHBO₃, 4 mM EDTA and 0.002%bromophenol blue, then electrophoresed on Criterion 5% or 10% TBE-ureadenaturing gels.

Example 11 Mutant T7 Polymerase

A. Background

A typical production and purification of mutant T7 RNA polymerase enzymeincludes the following:

-   -   1. Bacterial cell, BL21(DE3) chemically competent    -   2. Culture medium, LB    -   3. Antibiotics, ampicillin    -   4. Incubator with shaker    -   5. Centrifuge, Sorvall RC-5B or RC-5C (or equivalent)    -   6. FPLC system, GE/Amersham AKTA explorer (or new model AKTA        avant)    -   7. Ni-NTA affinity column, GE    -   8. Gel electrophoresis system, for example Invitrogen NuPage        apparatus and gels    -   9. Bench top microcentrifuge    -   10. Water bath or heating block    -   11. Pipet and others for transferring solution    -   12. Chemicals for buffers, normally from Sigma, VFW (molecular        biology grade)

The mutant T7 RNA polymerase may include two substitutions (Y639F andH784A) such as described in by Sousa (Padilla and Sousa, Nucl. AcidsRes. (2002) 30 (24):e138) and Ellington (Chelliserrykattil andEllington, Nature Biotechnology 22, 1155-1160 (2004)); hereinincorporated by reference in their entireties.

B. Transformation and Storage

For the purpose of transformation and storage of transformed bacterialcells cells are spread and grown on solid LB/agar plate containingappropriate antibiotics (ampicillin is the most commonly used),following the recommended protocol provided by manufacture forchemically competent cells, and incubated overnight at 37° C. Thebacterial colonies are grown on a solid surface and are stable for shortterm (1-4 weeks) storage only. For long term storage of transformedbacterial cells, a small scale liquid culture is grown in a singlecolony in 20 mL of LB broth containing antibiotics, where the cells havea final concentration of 15% glycerol and then freeze the cells inliquid nitrogen and store at −80° C. Optionally, an aliquot of purifiedplasmid DNA in high concentration is stored −80° C. for backup.

C. Purification

A culture in 20 mL LB broth containing 100 mg/mL of antibiotic isincubated at 37° C. overnight on a rotary shaker. The next day, 1 to 2ml of cells in culture medium is removed to inoculate 4 Liters of freshmedium. If needed these steps are repeated to increase the yield. Whenthe cells have grown to optical density (OD)600 of about 1.0 theexpression of T7 RNAP is induced with 1 mM isopropylb-D-thiogalactopyranoside (IPTG) or alternative promoter depending onthe expression vector. After the initiation of induction, the cells areallowed to grow for an additional three hours before they are harvestedby centrifugation at 3,600×g.

To preserve the activity of the enzyme all steps performed after thecells are harvested are carried out at 0-4° C. unless indicatedotherwise. The cell pellet from 4-8 L of culture is harvested in 1 Lcentrifuge bottles in a low speed centrifuge at 4° C. for 20-30 minutesand is re-suspended in 40-60 mL sonication buffer (50 mM sodiumphosphate, pH 8.0, 300 mM NaCl, 0.5 mM dithiothreitol (DTT), 20 mMimidazole) or the cell pellet is stored at −80° C. To disrupt cellsafter a 30-min incubation period at 4° C., protease inhibitors are addedto the cell suspension, the suspension is placed on ice andultrasonication is applied to the cell suspension for no more than 10seconds. To prevent overheating the cell suspension is placed on iceduring ultrasonication. The cell disruption step is repeating 3 timesbefore undergoing centrifugation at 10,000-12,000×g for 30 minutes toremove insoluble materials.

To break up the bacterial chromosomal DNA the crude extract is pressedtwice through a narrow syringe needle. The crude fraction is thenfiltered through a 0.2 uM Millpore filter and immediately applied onto a5-ml freshly nickel-chelated Ni-NTA affinity column with a flow rate of0.5-1 mL/minute and equilibrated with column buffer (50 mM sodiumphosphate, pH 8.0, 300 mM NaCl, 5% glycerol, 0.5 mM DTT, 20 mMimidazole) with a flow rate of 0.5 mL/min. The column is first washedwith at least 5 column volumes of column buffer to wash away unbound orloosely bound materials. The bound T7 RNA polymerase is eluted at 0.5ml/min by a linear imidazole gradient spanning from about 0.02 to 0.5 Min column buffer of 6 to 10 column volumes. The bound T7 RNA polymeraseis collected in 1 ml fractions in 96-well plates. Each fraction of boundT7 RNA polymerase within the single elution peak detectedspectrophotometrically at 280 nm is then examined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) at roomtemperature.

The crude fraction, flow through, and washing (collected as waste) isalso examined on SDS-PAGE gel at room temperature for QC purpose. Thefractions containing T7 RNA polymerase are pooled and dialyzed overnightagainst 1000 mL of storage buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mMDTT, 0.1 mM EDTA, 50% glycerol, 0.1% Triton X-100, pH 7.9 at 25° C.).The dialysis is repeated with new storage buffer for another 4 hoursbefore aliquots are prepared. If necessary, the overall volume isreduced by ultrafiltration to increase the enzyme concentration. Thepurified T7 RNA polymerase enzyme solution is stored at −20° C.

D. Concentration and Purity

The protein concentration in the final preparation is determinedaccording to the Bradford protein assay (Bio-Rad protein assay reagentand its method). The purity of T7 RNAP-muFA polymerase is measured bySDS-PAGE and stained by coomassie blue (for example, Pierce GelCode Bluestain reagent). The specific transcription activity of purified T7 RNApolymerase enzyme is measured using an in vitro transcription assay.

Example 12 In Vitro Transcription with Wild-Type T7 Polymerase

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 172; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 171; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) werefully modified with different chemistries and chemistry combinationslisted in Tables 12-15 using wild-type T7 polymerase as previouslydescribed.

The yield of the translation reactions was determined byspectrophometric measurement (0D260) and the yield for Luciferase isshown in Table 12 and G-CSF is shown in Table 14.The luciferase and G-CSF modified mRNA were also subjected to anenzymatic capping reaction and each modified mRNA capping reaction wasevaluated for yield by spectrophometic measurement (OD260) and correctsize assessed using bioanalyzer. The yield from the capping reaction forluciferase is shown in Table 13 and G-CSF is shown in Table 15.

TABLE 12 In vitro transcription chemistry for Luciferase Yield ChemicalModification (mg) N6-methyladenosine 0.99 5-methylcytidine 1.29N4-acetylcytidine 1.0 5-formylcytidine 0.55 Pseudouridine 2.0N1-methylpseudouridine 1.43 2-thiouridine 1.56 5-methoxyuridine 2.355-methyluridine 1.01 a-Thio-cytidine 0.83 5-Br-uridine (5Bru) 1.96 5 (2carbomethoxyvinyl) uridine 0.89 5 (3-1E propenyl Amino) uridine 2.01N4-acetylcytidine/pseudouridine 1.34N4-acetylcytidine/N1-methylpseudouridine 1.265-methylcytidine/5-methoxyuridine 1.38 5-methylcytidine/5-bromouridine0.12 5-methylcytidine/5-methyluridine 2.97 5-methylcytidine/half of theuridines are modified with 2-thiouridine 1.595-methylcytidine/2-thiouridine 0.90 5-methylcytidine/pseudouridine 1.835-methylcytidine/N1 methyl pseudouridine 1.33

TABLE 13 Capping chemistry and yield for Luciferase modified mRNA YieldChemical Modification (mg) 5-methylcytidine 1.02 N4-acetylcytidine 0.935-formylcytidine 0.55 Pseudouridine 2.07 N1-methylpseudouridine 1.272-thiouridine 1.44 5-methoxyuridine 2 5-methyluridine 0.8α-Thio-cytidine 0.74 5-Br-uridine (5Bru) 1.29 5 (2 carbomethoxyvinyl)uridine 0.54 5 (3-1E propenyl Amino) uridine 1.39N4-acetylcytidine/pseudouridine 0.99N4-acetylcytidine/N1-methylpseudouridine 1.085-methylcytidine/5-methoxyuridine 1.13 5-methylcytidine/5-methyluridine1.08 5-methylcytidine/half of the uridines are modified with2-thiouridine 1.2 5-methylcytidine/2-thiouridine 1.275-methylcytidine/pseudouridine 1.19 5-methylcytidine/N1 methylpseudouridine 1.04

TABLE 14 In vitro transcription chemistry and yield for G-CSF modifiedmRNA Yield Chemical Modification (mg) N6-methyladenosine 1.575-methylcytidine 2.05 N4-acetylcytidine 3.13 5-formylcytidine 1.41Pseudouridine 4.1 N1-methylpseudouridine 3.24 2-thiouridine 3.465-methoxyuridine 2.57 5-methyluridine 4.27 4-thiouridine 1.452′-F-uridine 0.96 α-Thio-cytidine 2.29 2′-F-guanosine 0.6N-1-methyladenosine 0.63 5-Br-uridine (5Bru) 1.08 5 (2carbomethoxyvinyl) uridine 1.8 5 (3-1E propenyl Amino) uridine 2.09N4-acetylcytidine/pseudouridine 1.72N4-acetylcytidine/N1-methylpseudouridine 1.375-methylcytidine/5-methoxyuridine 1.85 5-methylcytidine/5-methyluridine1.56 5-methylcytidine/half of the uridines are modified with2-thiouridine 1.84 5-methylcytidine/2-thiouridine 2.535-methylcytidine/pseudouridine 0.63 N4-acetylcytidine/2-thiouridine 1.3N4-acetylcytidine/5-bromouridine 1.37 5-methylcytidine/N1 methylpseudouridine 1.25 N4-acetylcytidine/pseudouridine 2.24

TABLE 15 Capping chemistry and yield for G-CSF modified mRNA ChemicalModification Yield (mg) N6-methyladenosine 1.04 5-methylcytidine 1.08N4-acetylcytidine 2.73 5-formylcytidine 0.95 Pseudouridine 3.88N1-methylpseudouridine 2.58 2-thiouridine 2.57 5-methoxyuridine 2.055-methyluridine 3.56 4-thiouridine 0.91 2′-F-uridine 0.54α-Thio-cytidine 1.79 2′-F-guanosine 0.14 5-Br-uridine (5Bru) 0.79 5 (2carbomethoxyvinyl) uridine 1.28 5 (3-1E propenyl Amino) uridine 1.78N4-acetylcytidine/pseudouridine 0.29N4-acetylcytidine/N1-methylpseudouridine 0.335-methylcytidine/5-methoxyuridine 0.91 5-methylcytidine/5-methyluridine0.61 5-methylcytidine/half of the uridines are modified 1.24 with2-thiouridine 5-methylcytidine/pseudouridine 1.08N4-acetylcytidine/2-thiouridine 1.34 N4-acetylcytidine/5-bromouridine1.22 5-methylcytidine/N1 methyl pseudouridine 1.56

Example 13 In Vitro Transcription with Mutant T7 Polymerase

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 172; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 171; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) werefully modified with different chemistries and chemistry combinationslisted in Tables 16-19 using a mutant T7 polymerase (Durascribe® T7Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.).

The yield of the translation reactions was determined byspectrophometric measurement (OD260) and the yield for Luciferase isshown in Table 16 and G-CSF is shown in Table 18.

The luciferase and G-CSF modified mRNA were also subjected to anenzymatic capping reaction and each modified mRNA capping reaction wasevaluated for yield by spectrophometic measurement (0D260) and correctsize assessed using bioanalyzer. The yield from the capping reaction forluciferase is shown in Table 17 and G-CSF is shown in Table 19.

TABLE 16 In vitro transcription chemistry and yield for Luciferasemodified mRNA Chemical Modification Yield (ug) 2′Fluorocytosine 71.42′Fluorouridine 57.5 5-methylcytosine/pseudouridine, test A 26.45-methylcytosine/N1-methylpseudouridine, test A 73.3N1-acetylcytidine/2-fluorouridine 202.2 5-methylcytidine/2-fluorouridine131.9 2-fluorocytosine/pseudouridine 119.32-fluorocytosine/N1-methylpseudouridine 107.02-fluorocytosine/2-thiouridine 34.7 2-fluorocytosine/5-bromouridine 81.02-fluorocytosine/2-fluorouridine 80.4 2-fluoroguanine/5-methylcytosine61.2 2-fluoroguanine/5-methylcytosine/pseudouridine 65.02-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 41.22-fluoroguanine/pseudouridine 79.12-fluoroguanine/N1-methylpseudouridine 74.65-methylcytidine/pseudouridine, test B 91.85-methylcytidine/N1-methylpseudouridine, test B 72.4 2′fluoroadenosine190.98

TABLE 17 Capping chemistry and yield for Luciferase modified mRNAChemical Modification Yield (ug) 2′Fluorocytosine 19.2 2′Fluorouridine16.7 5-methylcytosine/pseudouridine, test A 7.05-methylcytosine/N1-methylpseudouridine, test A 21.5Ni-acetylcytidine/2-fluorouridine 47.5 5-methylcytidine/2-fluorouridine53.2 2-fluorocytosine/pseudouridine 58.42-fluorocytosine/N1-methylpseudouridine 26.22-fluorocytosine/2-thiouridine 12.9 2-fluorocytosine/5-bromouridine 26.52-fluorocytosine/2-fluorouridine 35.7 2-fluoroguanine/5-methylcytosine24.7 2-fluoroguanine/5-methylcytosine/pseudouridine 32.32-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 31.32-fluoroguanine/pseudouridine 20.92-fluoroguanine/N1-methylpseudouridine 29.85-methylcytidine/pseudouridine, test B 58.25-methylcytidine/N1-methylpseudouridine, test B 44.4

TABLE 18 In vitro transcription chemistry and yield for G-CSF modifiedmRNA Chemical Modification Yield (ug) 2′Fluorocytosine 56.52′Fluorouridine 79.4 5-methylcytosine/pseudouridine, test A 21.25-methylcytosine/N1-methylpseudouridine, test A 77.1N1-acetylcytidine/2-fluorouridine 168.6 5-methylcytidine/2-fluorouridine134.7 2-fluorocytosine/pseudouridine 97.82-fluorocytosine/N1-methylpseudouridine 103.12-fluorocytosine/2-thiouridine 58.8 2-fluorocytosine/5-bromouridine 88.82-fluorocytosine/2-fluorouridine 93.9 2-fluoroguanine/5-methylcytosine97.3 2-fluoroguanine/5-methylcytosine/pseudouridine 96.02-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 82.02-fluoroguanine/pseudouridine 68.02-fluoroguanine/N1-methylpseudouridine 59.35-methylcytidine/pseudouridine, test B 58.75-methylcytidine/N1-methylpseudouridine, test B 78.0

TABLE 19 Capping chemistry and yield for G-CSF modified mRNA ChemicalModification Yield (ug) 2′Fluorocytosine 16.9 2′Fluorouridine 17.05-methylcytosine/pseudouridine, test A 10.65-methylcytosine/N1-methylpseudouridine, test A 22.7N1-acetylcytidine/2-fluorouridine 19.9 5-methylcytidine/2-fluorouridine21.3 2-fluorocytosine/pseudouridine 65.22-fluorocytosine/N1-methylpseudouridine 58.92-fluorocytosine/2-thiouridine 41.2 2-fluorocytosine/5-bromouridine 35.82-fluorocytosine/2-fluorouridine 36.7 2-fluoroguanine/5-methylcytosine36.6 2-fluoroguanine/5-methylcytosine/pseudouridine 37.32-fluoroguanine/5-methylcytidine/N1-methylpseudouridine 30.72-fluoroguanine/pseudouridine 29.02-fluoroguanine/N1-methylpseudouridine 22.75-methylcytidine/pseudouridine, test B 60.45-methylcytidine/N1-methylpseudouridine, test B 33.0

Example 14 2′O-methyl and 2′Fluoro Compounds

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 172; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) wereproduced as fully modified versions with the chemistries in Table 20 andtranscribed using mutant T7 polymerase (Durascribe® T7 Transcription kit(Cat. No. DS010925) (Epicentre®, Madison, Wis.). 2′ fluoro-containingmRNA were made using Durascribe T7, however, 2′Omethyl-containing mRNAcould not be transcribed using Durascribe T7.

Incorporation of 2′Omethyl modified mRNA might possibly be accomplishedusing other mutant T7 polymerases (Nat Biotechnol. (2004) 22:1155-1160;Nucleic Acids Res. (2002) 30:e138). Alternatively, 2′OMe modificationscould be introduced post-transcriptionally using enzymatic means.

Introduction of modifications on the 2′ group of the sugar has manypotential advantages. 2′OMe substitutions, like 2′ fluoro substitutionsare known to protect against nucleases and also have been shown toabolish innate immune recognition when incorporated into other nucleicacids such as siRNA and anti-sense (incorporated in its entirety,Crooke, ed. Antisense Drug Technology, 2^(nd) edition; Boca Raton: CRCpress).

The 2′Fluoro-modified mRNA were then transfected into HeLa cells toassess protein production in a cell context and the same mRNA were alsoassessed in a cell-free rabbit reticulocyte system. A control ofunmodified luciferase (natural luciferase) was used for bothtranscription experiments, a control of untreated and mock transfected(Lipofectamine 2000 alone) were also analyzed for the HeLa transfectionand a control of no RNA was analyzed for the rabbit reticulysates.

For the HeLa transfection experiments, the day before transfection,20,000 HeLa cells (ATCC no. CCL-2; Manassas, Va.) were harvested bytreatment with Trypsin-EDTA solution (LifeTechnologies, Grand Island,N.Y.) and seeded in a total volume of 100 ul EMEM medium (supplementedwith 10% FCS and 1× Glutamax) per well in a 96-well cell culture plate(Corning, Manassas, Va.). The cells were grown at 37oG in 5% CO₂atmosphere overnight. Next day, 83 ng of the 2′fluoro-containingluciferase modified RNA (mRNA sequence shown in SEQ ID NO: 172; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1) with the chemical modification described in Table 20, were dilutedin 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island,N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) wasused as transfection reagent and 0.2 ul were diluted in 10 ul finalvolume of OPTI-MEM. After 5 minutes of incubation at room temperature,both solutions were combined and incubated an additional 15 minute atroom temperature. Then the 20 ul combined solution was added to the 100ul cell culture medium containing the HeLa cells and incubated at roomtemperature. After 18 to 22 hours of incubation cells expressingluciferase were lysed with 100 ul of Passive Lysis Buffer (Promega,Madison, Wis.) according to manufacturer instructions. Aliquots of thelysates were transferred to white opaque polystyrene 96-well plates(Corning, Manassas, Va.) and combined with 100 ul complete luciferaseassay solution (Promega, Madison, Wis.). The lysate volumes wereadjusted or diluted until no more than 2 mio relative light units (RLU)per well were detected for the strongest signal producing samples andthe RLUs for each chemistry tested are shown in Table 20. The platereader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The backgroundsignal of the plates without reagent was about 200 relative light unitsper well.

For the rabbit reticulocyte lysate assay, 2′-fluoro-containingluciferase mRNA were diluted in sterile nuclease-free water to a finalamount of 250 ng in 10 ul and added to 40 ul of freshly prepared RabbitReticulocyte Lysate and the in vitro translation reaction was done in astandard 1.5 mL polypropylene reaction tube (Thermo Fisher Scientific,Waltham, Mass.) at 30° C. in a dry heating block. The translation assaywas done with the Rabbit Reticulocyte Lysate (nuclease-treated) kit(Promega, Madison, Wis.) according to the manufacturer's instructions.The reaction buffer was supplemented with a one-to-one blend of providedamino acid stock solutions devoid of either Leucine or Methionineresulting in a reaction mix containing sufficient amounts of both aminoacids to allow effective in vitro translation. After 60 minutes ofincubation, the reaction was stopped by placing the reaction tubes onice.

Aliquots of the in vitro translation reaction containing luciferasemodified RNA were transferred to white opaque polystyrene 96-well plates(Corning, Manassas, Va.) and combined with 100 ul complete luciferaseassay solution (Promega, Madison, Wis.). The volumes of the in vitrotranslation reactions were adjusted or diluted until no more than 2 miorelative light units (RLUs) per well were detected for the strongestsignal producing samples and the RLUs for each chemistry tested areshown in Table 21. The plate reader was a BioTek Synergy H1 (BioTek,Winooski, Vt.). The background signal of the plates without reagent wasabout 160 relative light units per well.

As can be seen in Table 20 and 21, multiple 2′Fluoro-containingcompounds are active in vitro and produce luciferase protein.

TABLE 20 HeLa Cells Chemical Concentration Volume Yield Modification(ug/ml) (ul) (ug) RLU 2′Fluoroadenosine 381.96 500 190.98 388.52′Fluorocytosine 654.56 500 327.28 2420 2′Fluoroguanine 541,795 500270.90 11,705.5 2′Flurorouridine 944.005 500 472.00 6767.5 Naturalluciferase N/A N/A N/A 133,853.5 Mock N/A N/A N/A 340 Untreated N/A N/AN/A 238

TABLE 21 Rabbit Reticulysates Chemical Modification RLU2′Fluoroadenosine 162 2′Fluorocytosine 208 2′Fluoroguanine 371,5092′Flurorouridine 258 Natural luciferase 2,159,968 No RNA 156

Example 15 Luciferase in HeLa Cells Using a Combination of Modifications

To evaluate using of 2′fluoro-modified mRNA in combination with othermodification a series of mRNA were transcribed using either wild-type T7polymerase (non-fluoro-containing compounds) or using mutant T7polymerases (fluyoro-containing compounds) as described in Example 14.All modified mRNA were tested by in vitro transfection in HeLa cells.

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 ul EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37oG in 5% CO₂ atmosphere overnight. Next day, 83 ng ofLuciferase modified RNA (mRNA sequence shown in SEQ ID NO: 172; polyAtail of approximately 160 nucleotides not shown in sequence; 5′cap,Cap1) with the chemical modification described in Table 22, were dilutedin 10 ul final volume of OPTI-MEM (LifeTechnologies, Grand Island,N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) wasused as transfection reagent and 0.2 ul were diluted in 10 ul finalvolume of OPTI-MEM. After 5 minutes of incubation at room temperature,both solutions were combined and incubated an additional 15 minute atroom temperature. Then the 20 ul combined solution was added to the 100ul cell culture medium containing the HeLa cells and incubated at roomtemperature.

After 18 to 22 hours of incubation cells expressing luciferase werelysed with 100 ul of Passive Lysis Buffer (Promega, Madison, Wis.)according to manufacturer instructions. Aliquots of the lysates weretransferred to white opaque polystyrene 96-well plates (Corning,Manassas, Va.) and combined with 100 ul complete luciferase assaysolution (Promega, Madison, Wis.). The lysate volumes were adjusted ordiluted until no more than 2 mio relative light units (RLU) per wellwere detected for the strongest signal producing samples and the RLUsfor each chemistry tested are shown in Table 22. The plate reader was aBioTek Synergy H1 (BioTek, Winooski, Vt.). The background signal of theplates without reagent was about 200 relative light units per well.

As evidenced in Table 22, most combinations of modifications resulted inmRNA which produced functional luciferase protein, including all thenon-flouro containing compounds and many of the combinations containing2′fluro modifications.

TABLE 22 Luciferase Chemical Modification RLUN4-acetylcytidine/pseudouridine 113,796N4-acetylcytidine/N1-methylpseudouridine 316,3265-methylcytidine/5-methoxyuridine 24,9485-methylcytidine/5-methyluridine 43,675 5-methylcytidine/half of theuridines modified with 41,601 50% 2-thiouridine5-methylcytidine/2-thiouridine 1,102 5-methylcytidine/pseudouridine51,035 5-methylcytidine/N1 methyl pseudouridine 152,151N4-acetylcytidine/2′Fluorouridine triphosphate 2885-methylcytidine/2′Fluorouridine triphosphate 269 2′Fluorocytosinetriphosphate/pseudouridine 260 2′Fluorocytosinetriphosphate/N1-methylpseudouridine 412 2′Fluorocytosinetriphosphate/2-thiouridine 427 2′Fluorocytosinetriphosphate/5-bromouridine 253 2′Fluorocytosinetriphosphate/2′Fluorouridine triphosphate 184 2′Fluoroguaninetriphosphate/5-methylcytidine 321 2′Fluoroguaninetriphosphate/5-methylcytidine/Pseudouridine 2072′Fluoroguanine/5-methylcytidine/N1 methylpsuedouridine 2352′Fluoroguanine/pseudouridine 218 2′Fluoroguanine/N1-methylpsuedouridine247 5-methylcytidine/pseudouridine, test A 13,8335-methylcytidine/N-methylpseudouridine, test A 598 2′Fluorocytosinetriphosphate 201 2′Fluorouridine triphosphate 3055-methylcytidine/pseudouridine, test B 115,4015-methylcytidine/N-methylpseudouridine, test B 21,034 Natural luciferase30,801 Untreated 344 Mock 262

Example 16 G-CSF In Vitro Transcription

To assess the activity of all our different chemical modifications inthe context of a second open reading frame, we replicated experimentspreviously conducted using luciferase mRNA, with human G-CSF mRNA. G-CSFmRNA (mRNA sequence shown in SEQ ID NO: 171; polyA tail of approximately160 nucleotides not shown in sequence; 5′cap, Cap1) were fully modifiedwith the chemistries in Tables 23 and 24 using wild-type T7 polymerase(for all non-fluoro-containing compounds) or mutant T7 polymerase (forall fluoro-containing compounds). The mutant T7 polymerase was obtainedcommercially (Durascribe® T7 Transcription kit (Cat. No. DS010925)(Epicentre®, Madison, Wis.).

The modified RNA in Tables 23 and 24 were transfected in vitro in HeLacells or added to rabbit reticulysates (250 ng of modified mRNA) asindicated. A control of untreated, mock transfected (transfectionreagent alone), G-CSF fully modified with 5-methylcytosine andN1-methylpseudouridine or luciferase control (mRNA sequence shown in SEQID NO: 172; polyA tail of approximately 160 nucleotides not shown insequence; 5′cap, Cap1) fully modified with 5-methylcytosine andN1-methylpseudouridine were also analyzed. The expression of G-CSFprotein was determined by ELISA and the values are shown in Tables 23and 24. In Table 23, “NT” means not tested.

As shown in Table 23, many, but not all, chemical modifications resultedin human G-CSF protein production. These results from cell-based andcell-free translation systems correlate very nicely with the samemodifications generally working or not working in both systems. Onenotable exception is 5-formylcytidine modified G-CSF mRNA which workedin the cell-free translation system, but not in the HeLa cell-basedtransfection system. A similar difference between the two assays wasalso seen with 5-formylcytidine modified luciferase mRNA.

As demonstrated in Table 24, many, but not all, G-CSF mRNA modifiedchemistries (when used in combination) demonstrated in vivo activity. Inaddition the presence of N1-methylpseudouridine in the modified mRNA(with N4-acetylcytidine or 5 methylcytidine) demonstrated higherexpression than when the same combinations where tested using withpseudouridine. Taken together, these data demonstrate thatN1-methylpseudouridine containing G-CSF mRNA results in improved proteinexpression in vitro.

TABLE 23 G-CSF Expression G-CSF protein G-CSF (pg/ml) protein Rabbit(pg/ml) reticulysates Chemical Modification HeLa cells cellsPseudouridine 1,150,909 147,875 5-methyluridine 347,045 147,2502-thiouridine 417,273 18,375 N1-methylpseudouridine NT 230,0004-thiouridine 107,273 52,375 5-methoxyuridine 1,715,909 201,7505-methylcytosine/pseudouridine, Test A 609,545 119,7505-methylcytosine/N1-methylpseudouridine, Test A 1,534,318 110,5002′-Fluoro-guanosine 11,818 0 2′-Fluoro-uridine 60,455 05-methylcytosine/pseudouridine, Test B 358,182 57,8755-methylcytosine/N1-methylpseudouridine, Test B 1,568,636 76,7505-Bromo-uridine 186,591 72,000 5-(2carbomethoxyvinyl) uridine 1,364 05-[3(1-E-propenylamino) uridine 27,955 32,625 α-thio-cytidine 120,45542,625 5-methylcytosine/pseudouridine, Test C 882,500 49,250Nl-methyl-adenosine 4,773 0 N6-methyl-adenosine 1,591 05-methyl-cytidine 646,591 79,375 N4-acetylcytidine 39,545 8,0005-formyl-cytidine 0 24,000 5-methylcytosine/pseudouridine, Test D 87,04547,750 5-methylcytosine/N1-methylpseudouridine, Test D 1,168,864 97,125Mock 909 682 Untreated 0 0 5-methylcytosine/N1-methylpseudouridine,Control 1,106,591 NT Luciferase control NT 0

TABLE 24 Combination Chemistries in HeLa cells G-CSF protein (pg/ml)Chemical Modification HeLa cells N4-acetylcytidine/pseudouridine 537,273N4-acetylcytidine/N1-methylpseudouridine 1,091,8185-methylcytidine/5-methoxyuridine 516,1365-methylcytidine/5-bromouridine 48,864 5-methylcytidine/5-methyluridine207,500 5-methylcytidine/2-thiouridine 33,409N4-acetylcytidine/5-bromouridine 211,591 N4-acetylcytidine/2-thiouridine46,136 5-methylcytosine/pseudouridine 301,3645-methylcytosine/N1-methylpseudouridine 1,017,727N4-acetylcytidine/2′Fluorouridine triphosphate 62,2735-methylcytidine/2′Fluorouridine triphosphate 49,318 2′Fluorocytosinetriphosphate/pseudouridine 7,955 2′Fluorocytosinetriphosphate/N1-methylpseudouridine 1,364 2′Fluorocytosinetriphosphate/2-thiouridine 0 2′Fluorocytosinetriphosphate/5-bromouridine 1,818 2′Fluorocytosinetriphosphate/2′Fluorouridine triphosphate 909 2′Fluoroguaninetriphosphate/5-methylcytidine 0 2′Fluoroguaninetriphosphate/5-methylcytidine/pseudouridine 0 2′Fluoroguaninetriphosphate/5-methylcytidine/N1 1,818 methylpseudouridine2′Fluoroguanine triphosphate/pseudouridine 1,136 2′Fluoroguaninetriphosphate/2′Fluorocytosine 0 triphosphate/Nl-methylpseudouridine5-methylcytidine/pseudouridine 617,7275-methylcytidine/N1-methylpseudouridine 747,0455-methylcytidine/pseudouridine 475,4555-methylcytidine/N1-methylpseudouridine 689,0915-methylcytosine/N1-methylpseudouridine, Control 1 848,4095-methylcytosine/N1-methylpseudouridine, Control 2 581,818 Mock 682Untreated 0 Luciferase 2′Fluorocytosine triphosphate 0 Luciferase2′Fluorouridine triphosphate 0

Example 17 Screening of Chemistries

The tables listed in below (Tables 25-27) summarize much of the in vitroand in vitro screening data with the different compounds presented inthe previous examples. A good correlation exists between cell-based andcell-free translation assays. The same chemistry substitutions generallyshow good concordance whether tested in the context of luciferase orG-CSF mRNA. Lastly, N1-methylpseudouridine containing mRNA show a veryhigh level of protein expression with little to no detectable cytokinestimulation in vitro and in vivo, and is superior to mRNA containingpseudouridine both in vitro and in vivo.

Luciferase mRNA (mRNA sequence shown in SEQ ID NO: 172; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) andG-CSF mRNA (mRNA sequence shown in SEQ ID NO: 171; polyA tail ofapproximately 160 nucleotides not shown in sequence; 5′cap, Cap1) weremodified with naturally and non-naturally occurring chemistriesdescribed in Tables 25 and 26 or combination chemistries described inTable 27 and tested using methods described herein.

In Tables 25 and 26, “*” refers to in vitro transcription reaction usinga mutant T7 polymerase (Durascribe® T7 Transcription kit (Cat. No.DS010925) (Epicentre®, Madison, Wis.); “* *” refers to the second resultin vitro transcription reaction using a mutant T7 polymerase(Durascribe® T7 Transcription kit (Cat. No. DS010925) (Epicentre®,Madison, Wis.); “***” refers to production seen in cell freetranslations (rabbit reticulocyte lysates); the protein production ofHeLa is judged by “+,” “+/−” and “−”; when referring to G-CSF PBMC“++++” means greater than 6,000 pg/ml G-CSF, “+++” means greater than3,000 pg/ml G-CSF, “++” means greater than 1,500 pg/ml G-CSF, “+” meansgreater than 300 pg/ml G-CSF, “+/−” means 150-300 pg/ml G-CSF and thebackground was about 110 pg/ml; when referring to cytokine PBMC “++++”means greater than 1,000 pg/ml interferon-alpha (IFN-alpha), “+++” meansgreater than 600 pg/ml IFN-alpha, “++” means greater than 300 pg/mlIFN-alpha, “+” means greater than 100 pg/ml IFN-alpha, “−” means lessthan 100 pg/ml and the background was about 70 pg/ml; and “NT” means nottested. In Table 27, the protein production was evaluated using a mutantT7 polymerase (Durascribe® T7 Transcription kit (Cat. No. DS010925)(Epicentre®, Madison, Wis.).

TABLE 25 Naturally Occurring In Protein Protein Cytokines In Vivo IVTProtein (G- (G- (G- Vivo Protein Common Name IVT (G- (Luc; CSF; CSF;CSF; Protein (G- (symbol) (Luc) CSF) HeLa) HeLa) PBMC) PBMC) (Luc) CSF)1-methyladenosine Fail Pass NT − +/− ++ NT NT (m¹A) N⁶- Pass Pass − −+/− ++++ NT NT methyladenosine (m⁶A) 2′-O- Fail* Not NT NT NT NT NT NTmethyladenosine Done (Am) 5-methylcytidine Pass Pass + + + ++ + NT (m⁵C)2′-O- Fail* Not NT NT NT NT NT NT methylcytidine Done (Cm)2-thiocytidine Fail Fail NT NT NT NT NT NT (s²C) N⁴-acetylcytidine PassPass + + +/− +++ + NT (ac⁴C) 5-formylcytidine Pass Pass −*** −*** − + NTNT (f⁵C) 2′-O- Fail* Not NT NT NT NT NT NT methylguanosine Done (Gm)inosine (I) Fail Fail NT NT NT NT NT NT pseudouridine (Y) Pass Pass + +++ + + NT 5-methyluridine Pass Pass + + +/− + NT NT (m⁵U)2′-O-methyluridine Fail* Not NT NT NT NT NT NT (Um) Done 1- Pass Pass +Not ++++ − + NT methylpseudouridine Done (m¹Y) 2-thiouridine (s²U) PassPass − + + + NT NT 4-thiouridine (s⁴U) Fail Pass + +/− ++ NT NT5-methoxyuridine Pass Pass + + ++ − + NT (mo⁵U) 3-methyluridine FailFail NT NT NT NT NT NT (m³U)

TABLE 26 Non-Naturally Occurring In Protein Protein Cytokines In VivoIVT Protein (G- (G- (G- Vivo Protein Common IVT (G- (Luc; CSF; CSF; CSF;Protein (G- Name (Luc) CSF) HeLa) HeLa) PBMC) PBMC) (Luc) CSF) 2′-F-ara-Fail Fail NT NT NT NT NT NT guanosine 2′-F-ara- Fail Fail NT NT NT NT NTNT adenosine 2′-F-ara- Fail Fail NT NT NT NT NT NT cytidine 2′-F-ara-Fail Fail NT NT NT NT NT NT uridine 2′-F-guanosine Fail/ Pass/ +** +/−− + + NT Pass Fail ** ** 2′-F-adenosine Fail/ Fail/ −** NT NT NT NT NTPass** Fail** 2′-F-cytidine Fail/ Fail/ +** NT NT NT + NT Pass Pass **** 2′-F-uridine Fail/ Pass/ +** + +/− + − NT Pass Pass ** ** 2′-OH-ara-Fail Fail NT NT NT NT NT NT guanosine 2′-OH-ara- Not Not NT NT NT NT NTNT adenosine Done Done 2′-OH-ara- Fail Fail NT NT NT NT NT NT cytidine2′-OH-ara- Fail Fail NT NT NT NT NT NT uridine 5-Br-Uridine PassPass + + + + + 5-(2- Pass Pass − − +/− − carbomethoxyvinyl) Uridine5-[3-(1-E- Pass Pass − + + − Propenylamino) Uridine (aka Chem 5) N6-(19-Fail Fail NT NT NT NT NT NT Amino- pentaoxanonadecyl) A 2- Fail Fail NTNT NT NT NT NT Dimethylamino guanosine 6-Aza-cytidine Fail Fail NT NT NTNT NT NT a-Thio- Pass Pass + + +/− +++ NT NT cytidine Pseudo- NT NT NTNT NT NT NT NT isocytidine 5-Iodo-uridine NT NT NT NT NT NT NT NTa-Thio-uridine NT NT NT NT NT NT NT NT 6-Aza-uridine NT NT NT NT NT NTNT NT Deoxy- NT NT NT NT NT NT NT NT thymidine a-Thio NT NT NT NT NT NTNT NT guanosine 8-Oxo- NT NT NT NT NT NT NT NT guanosine O6-Methyl- NTNT NT NT NT NT NT NT guanosine 7-Deaza- NT NT NT NT NT NT NT NTguanosine 6-Chloro- NT NT NT NT NT NT NT NT purine a-Thio- NT NT NT NTNT NT NT NT adenosine 7-Deaza- NT NT NT NT NT NT NT NT adenosine 5-iodo-NT NT NT NT NT NT NT NT cytidine

In Table 27, the protein production of HeLa is judged by “+,” “+1-” and“−”; when referring to G-CSF PBMC “++++” means greater than 6,000 pg/mlG-CSF, “+++” means greater than 3,000 pg/ml G-CSF, “++” means greaterthan 1,500 pg/ml G-CSF, “+” means greater than 300 pg/ml G-CSF, “+/−”means 150-300 pg/ml G-CSF and the background was about 110 pg/ml; whenreferring to cytokine PBMC “++++” means greater than 1,000 pg/mlinterferon-alpha (IFN-alpha), “+++” means greater than 600 pg/mlIFN-alpha, “++” means greater than 300 pg/ml IFN-alpha, “+” meansgreater than 100 pg/ml IFN-alpha, “−” means less than 100 pg/ml and thebackground was about 70 pg/ml; “WT” refers to the wild type T7polymerase, “MT” refers to mutant T7 polymerase (Durascribe® T7Transcription kit (Cat. No. DS010925) (Epicentre®, Madison, Wis.) and“NT” means not tested.

TABLE 27 Combination Chemistry Protein Protein In IVT Protein (G- (G-Cytokines Vivo Cytidine Uridine IVT (G- (Luc; CSF; CSF; (G-CSF; Proteinanalog analog Purine Luc CSF) HeLa) HeLa) PBMC) PBMC) (Luc) N4-pseudouridine A, G Pass Pass + + NT NT + acetylcytidine WT WT N4- N1- A,G Pass Pass + + NT NT + acetylcytidine methylpseudouridine WT WT 5- 5-A, G Pass Pass + + NT NT + methylcytidine methoxyuridine WT WT 5- 5- A,G Pass Pass Not + NT NT methylcytidine bromouridine WT WT Done 5- 5- A,G Pass Pass + + NT NT + methylcytidine methyluridine WT WT 5- 50% 2- A,G Pass Pass + NT NT NT + methylcytidine thiouridine; WT WT 50% uridine5- 100% 2- A, G Pass Pass − + NT NT methylcytidine thiouridine WT WT 5-pseudouridine A, G Pass Pass + + ++ + + methylcytidine WT WT 5- N1- A, GPass Pass + + ++++ − + methylcytidine methylpseudouridine WT WT N4- 2-A, G Not Pass Not + NT NT NT acetylcytidine thiouridine Done WT Done N4-5- A, G Not Pass Not + NT NT NT acetylcytidine bromouridine Done WT DoneN4- 2 A, G Pass Pass − + NT NT NT acetylcytidine Fluorouridinetriphosphate 5- 2 A, G Pass Pass − + NT NT NT methylcytidineFluorouridine triphosphate 2 pseudouridine A, G Pass Pass − + NT NT NTFluorocytosine triphosphate 2 N1- A, G Pass Pass − +/− NT NT NTFluorocytosine methylpseudouridine triphosphate 2 2- A, G Pass Pass − −NT NT NT Fluorocytosine thiouridine triphosphate 2 5- A, G Pass Pass −+/− NT NT NT Fluorocytosine bromouridine triphosphate 2 2 A, G Pass Pass− +/− NT NT NT Fluorocytosine Fluorouridine triphosphate triphosphate 5-uridine A, 2 Pass Pass − − NT NT NT methylcytidine Fluoro GTP 5-pseudouridine A, 2 Pass Pass − − NT NT NT methylcytidine Fluoro GTP 5-N1- A, 2 Pass Pass − +/− NT NT NT methylcytidine methylpseudouridineFluoro GTP 2 pseudouridine A, 2 Pass Pass − +/− NT NT NT FluorocytosineFluoro triphosphate GTP 2 N1- A, 2 Pass Pass − − NT NT NT Fluorocytosinemethylpseudouridine Fluoro triphosphate GTP

Example 18 Polymerases for Making mRNA

A. D4 RNA Polymerase Derived from Tgo

D4 RNA polymerase is derived from Tgo, the replicative DNA polymerasefrom the hyperthermophillic archaeon Thermococcus gorgonarius, isproduced by the method described in International Patent Publication NoWO2011135280 (See e.g., Examples 7, 8, 9 and 25) and/or by the methoddescribed by Cozens et al. (A short adaptive path from DNA to RNApolymerases. PNAS 2012:109(21) 8067-8072), each of which is hereinincorporated by reference in its entirety. The D4 RNA polymerase may beused in making the primary constructs, polynucleotides and/or mmRNAdescribed here using the methods known in the art and/or describedherein. The primary constructs, polynucleotides and/or mmRNA can includevarious chemical modifications described herein.

B. Thermostable RNA Polymerase

A thermostable RNA polymerase, TNQ, derived from Tgo comprising themutations Y409N and E664Q is produced by the methods described inInternational Patent Publication No WO2011135280 and U.S. PatentPublication No US20130130320, each of which is herein incorporated byreference in its entirety (see e.g., Example 25 of WO2011135280 orUS20130130320). Primary constructs, polynucleotides and/or mmRNA of thepresent invention are synthesized using the TNQ polymerase. The primaryconstructs, polynucleotides and/or mmRNA can include various chemicalmodifications described herein. Methods of making mRNA are known in theart, described herein and/or described in International PatentPublication No WO2011135280 and U.S. Patent Application US20130130320(see e.g., Examples 11 and 25), each of whch is herein incorporated byreference in its entirety.

C. Screening Mutations for Optimal RNA Synthesis

Different mutations of DNA polymerases can be screened in order todetermine the optimal mutations for RNA synthesis. In InternationalPatent Publication No WO2011135280 and U.S. Patent ApplicationUS20130130320, each of which herein incorporated by reference in itsentirety, a single point mutation in the 10A region (E664Q) of Tgo wasfound to be necessary for processive RNA synthesis. To determine ifother mutations in this critical area would affect RNA synthesismutations at position 664 and at the positions prior and after position664 are mutated and analyzed for their ability to allow RNA synthesis(see e.g., Example 12 of International Patent Publication NoWO2011135280 or U.S. Patent Application US20130130320, each of which isherein incorporated by reference in its entirety)

Example 19 Fusion Enzyme for Synthesizing and Capping mRNA

A recombinant fusion (chimeric) enzyme for synthesizing and capping mRNAis designed, expressed, purified and used for in vitro transcription andcapping.

In one embodiment, the methods used are those described in U.S. PatentPublication No. US20130042334 and International Patent Publication NoWO2011128444, each of which is herein incorporated by reference in itsentirety, herein incorporated by reference in its entirety.

In one embodiment, an mRNA is synthesized using a recombinantpolymerase-capping fusion enzyme. The fusion enzyme includes both a RNApolymerase (RNAP) enzyme and a capping enzyme. The RNAP can be a T7RNAP.

Other possible RNAPs include T3 RNAP or SP6 RNAP. The capping enzyme canbe a Vaccinia capping enzyme, e.g., the catalytic (D1) and stimulation(D12) subunits. Alternatively, the capping enzyme is a single subunitcapping enzyme. Nucleic acid and amino acid sequences of representativeT7 RNAP and Vaccinia capping enzymes are shown below.

In one embodiment, the fusion enzyme includes a 2 subunit cappingenzyme. The fusion enzyme can be expressed as a single polypeptide thatincludes both capping enzyme subunits and the RNAP. Alternatively, thefusion enzyme can be expressed as two polypeptides, each having one ofthe capping enzyme subunits and one polypeptide also having the RNAP. Inboth embodiments (one or two polypeptides), the fusion enzyme is capableof in vivo or in vitro assembly into a functional fusion enzyme. It hasbeen shown that the solubility of the catalytic subunit of a Vacciniacapping enzyme is improved by co-expression with the smaller stimulationsubunit. Co expression with a RNAP, e.g., T7 RNA polymerase, willfurther improve the solubility.

Both wild type and mutant version of the RNAP and capping enzymesequences are contemplated. Direct mutations or molecular evolution canbe used to create mutant versions of the enzyme useful for, e.g.,incorporation of modified nucleotides.

The fusion enzyme can include linkers between the RNAP and (one or bothsubunits of) the capping enzyme. Linker sequences are well known to oneof skill in the art and typically have the following structure: (GGGGS)nwhere n can be any whole number. Exemplary linker sequences include thesequence in Table 28.

TABLE 28 Linker Sequences Sequence SEQ ID NO LGGGGSGGGGSGGGGSAAA 173LSGGGGSGGGGSGGGGSGGGGSAAA 174 EGKSSGSGSESKST 175

The fusion enzyme may include affinity tag sequences useful for, e.g.,purification. Examples include a His tag for Nickel based purification.

The open reading frame encoding the fusion enzyme (one or morepolypeptides, wild-type or mutant, with or without linkers, with orwithout an affinity tag) will be chemically synthesized, inserted intothe expression vector and verified by DNA sequencing. In someembodiments, the sequence will be codon optimized for, e.g., improvedprotein synthesis/yield in bacterial, yeast or mammalian cells.

The fusion enzyme (one or more polypeptides, with or without linkers,with or without an affinity tag) is expressed in, e.g., a bacterialexpression system. In some embodiments, the fusion enzyme is expressedusing, e.g., a pEXpress vector from DNA2.0 in the expression host cellBL21(DE3) from Invitrogen. Alternatively the pET system from Invitrogenis used to express the fusion enzyme. Other expression systems are wellknown in the art. In some embodiments a lower cell growth temperature isused to increase solubility of the fusion enzyme.

The fusion enzyme is purified using methods known to one of skill in theart using, e.g., published protocols for RNAP and/or capping enzymes.The purification can include, e.g., ion exchange chromatography, sizeexclusion chromatography, and/or Ni-NTA affinity chromatography.

The purified fusion enzyme is used for a one-step enzymatic reactionthat combines mRNA synthesis and 5′ capping of mRNA using the methodsdescribed herein. The reaction buffer includes some or all of thefollowing components:

-   -   Buffer, e.g., Tris-HCl to control the pH of the reaction mix    -   Mg and Mn for the optimal enzymatic activity and specificity    -   Single nucleoside triphosphates (modified or not natural) of        desired combination (composition)    -   Spermidine    -   RNase inhibitor    -   Pyrophosphaste    -   SAM (methyl donor)

Example 20 Vaccinia Capping Enzyme and T7 RNA Polymerase Chimeric Enzyme

A bacterial plasmid encoding a fusion enzyme of Vaccinia capping enzyme(D1 and D12) and T7 RNA polymerase can be produced by the methodsdescribed herein, known in the art and/or described in U.S. PatentPublication No. US20130042334 and International Patent Publication NoWO2011128444, each of which is herein incorporated by reference in itsentirety, herein incorporated by reference in its entirety. Thebacterial plasmid can be a plasmid known in the art or a bacterialplasmid which has been modified to synthesize the chimeric enzyme. Thechimeric enzyme can be purified before it is used to synthesize and capthe mRNAs.

Example 21 Capping Enzymes and RNA Polymerase Sequences

Capping enzymes and RNA polymerases are used to synthesize and cap mRNA.In one embodiment, the capping enzymes comprise a Vaccinia cappingenzyme such as, but not limited to, a Vaccinia capping enzyme comprisinga D1 subunit and a D12 subunit or comprising a single subunit cappingenzyme. In another embodiment, the RNA polymerase is a T7 RNApolymerase, T3 RNA polymerase a SP6 RNA polymerase, a RNA polymerasevariant and a DNA polymerase mutant. A non-exhaustive listing ofsequences of capping enzymes and T7 RNA polymerases are described inTable 29.

TABLE 29 Capping Enzymes and RNA Polymerase Sequences SEQ ID DescriptionSequence NO T7 RNA ATGAACACGATTAACATCGCTAAGAACGACTTCTCTGACATCGAA 176Polymerase CTGGCTGCTATCCCGTTCAACACTCTGGCTGACCATTACGGTGAGC (Genbank IDGTTTAGCTCGCGAACAGTTGGCCCTTGAGCATGAGTCTTACGAGAT ACY75835.1;GGGTGAAGCACGCTTCCGCAAGATGTTTGAGCGTCAACTTAAAGC DNATGGTGAGGTTGCGGATAACGCTGCCGCCAAGCCTCTCATCACTACC Sequence)CTACTCCCTAAGATGATTGCACGCATCAACGACTGGTTTGAGGAAGTGAAAGCTAAGCGCGGCAAGCGCCCGACAGCCTTCCAGTTCCTGCAAGAAATCAAGCCGGAAGCCGTAGCGTACATCACCATTAAGACCACTCTGGCTTGCCTAACCAGTGCTGACAATACAACCGTTCAGGCTGTAGCAAGCGCAATCGGTCGGGCCATTGAGGACGAGGCTCGCTTCGGTCGTATCCGTGACCTTGAAGCTAAGCACTTCAAGAAAAACGTTGAGGAACAACTCAACAAGCGCGTAGGGCACGTCTACAAGAAAGCATTTATGCAAGTTGTCGAGGCTGACATGCTCTCTAAGGGTCTACTCGGTGGCGAGGCGTGGTCTTCGTGGCATAAGGAAGACTCTATTCATGTAGGAGTACGCTGCATCGAGATGCTCATTGAGTCAACCGGAATGGTTAGCTTACACCGCCAAAATGCTGGCGTAGTAGGTCAAGACTCTGAGACTATCGAACTCGCACCTGAATACGCTGAGGCTATCGCAACCCGTGCAGGTGCGCTGGCTGGCATCTCTCCGATGTTCCAACCTTGCGTAGTTCCTCCTAAGCCGTGGACTGGCATTACTGGTGGTGGCTATTGGGCTAACGGTCGTCGTCCTCTGGCGCTGGTGCGTACTCACAGTAAGAAAGCACTGATGCGCTACGAAGACGTTTACATGCCTGAGGTGTACAAAGCGATTAACATTGCGCAAAACACCGCATGGAAAATCAACAAGAAAGTCCTAGCGGTCGCCAACGTAATCACCAAGTGGAAGCATTGTCCGGTCGAGGACATCCCTGCGATTGAGCGTGAAGAACTCCCGATGAAACCGGAAGACATCGACATGAATCCTGAGGCTCTCACCGCGTGGAAACGTGCTGCCGCTGCTGTGTACCGCAAGGACAAGGCTCGCAAGTCTCGCCGTATCAGCCTTGAGTTCATGCTTGAGCAAGCCAATAAGTTTGCTAACCATAAGGCCATCTGGTTCCCTTACAACATGGACTGGCGCGGTCGTGTTTACGCTGTGTCAATGTTCAACCCGCAAGGTAACGATATGACCAAAGGACTGCTTACGCTGGCGAAAGGTAAACCAATCGGTAAGGAAGGTTACTACTGGCTGAAAATCCACGGTGCAAACTGTGCGGGTGTCGATAAGGTTCCGTTCCCTGAGCGCATCAAGTTCATTGAGGAAAACCACGAGAACATCATGGCTTGCGCTAAGTCTCCACTGGAGAACACTTGGTGGGCTGAGCAAGATTCTCCGTTCTGCTTCCTTGCGTTCTGCTTTGAGTACGCTGGGGTACAGCACCACGGCCTGAGCTATAACTGCTCCCTTCCGCTGGCGTTTGACGGGTCTTGCTCTGGCATCCAGCACTTCTCCGCGATGCTCCGAGATGAGGTAGGTGGTCGCGCGGTTAACTTGCTTCCTAGTGAAACCGTTCAGGACATCTACGGGATTGTTGCTAAGAAAGTCAACGAGATTCTACAAGCAGACGCAATCAATGGGACCGATAACGAAGTAGTTACCGTGACCGATGAGAACACTGGTGAAATCTCTGAGAAAGTCAAGCTGGGCACTAAGGCACTGGCTGGTCAATGGCTGGCTTACGGTGTTACTCGCAGTGTGACTAAGCGTTCAGTCATGACGCTGGCTTACGGGTCCAAAGAGTTCGGCTTCCGTCAACAAGTGCTGGAAGATACCATTCAGCCAGCTATTGATTCCGGCAAGGGTCTGATGTTCACTCAGCCGAATCAGGCTGCTGGATACATGGCTAAGCTGATTTGGGAATCTGTGAGCGTGACGGTGGTAGCTGCGGTTGAAGCAATGAACTGGCTTAAGTCTGCTGCTAAGCTGCTGGCTGCTGAGGTCAAAGATAAGAAGACTGGAGAGATTCTTCGCAAGCGTTGCGCTGTGCATTGGGTAACTCCTGATGGTTTCCCTGTGTGGCAGGAATACAAGAAGCCTATTCAGACGCGCTTGAACCTGATGTTCCTCGGTCAGTTCCGCTTACAGCCTACCATTAACACCAACAAAGATAGCGAGATTGATGCACACAAACAGGAGTCTGGTATCGCTCCTAACTTTGTACACAGCCAAGACGGTAGCCACCTTCGTAAGACTGTAGTGTGGGCACACGAGAAGTACGGAATCGAATCTTTTGCACTGATTCACGACTCCTTCGGTACCATTCCGGCTGACGCTGCGAACCTGTTCAAAGCAGTGCGCGAAACTATGGTTGACACATATGAGTCTTGTGATGTACTGGCTGATTTCTACGACCAGTTCGCTGACCAGTTGCACGAGTCTCAATTGGACAAAATGCCAGCACTTCCGGCTAAAGGTAACTTGAACCTCCGTGACATCTTAGA GTCGGACTTCGCGTTCGCGTAA T7RNA MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGE 177 PolymeraseARFRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAK (Genbank IDRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRA ACY75835.1;IEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADM ProteinLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVV Sequence)GQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA VacciniaATGGATGCCAACGTAGTATCATCTTCTACTATTGCGACGTATATAG 178 cappingACGCTTTAGCGAAGAATGCTTCGGAATTAGAACAGAGGTCTACCG enzymeCATACGAAATAAATAATGAATTGGAACTAGTATTTATTAAGCCGC catalyticCATTGATTACTTTGACAAATGTAGTGAATATCTCTACGATTCAGGA subunit (D1):ATCGTTTATTCGATTTACCGTTACTAATAAGGAAGGTGTTAAAATT GenBankAGAACTAAGATTCCATTATCTAAGGTACATGGTCTAGATGTAAAA M15058.1:AATGTACAGTTAGTAGATGCTATAGATAACATAGTTTGGGAAAAG Vaccinia virusAAATCATTAGTGACGGAAAATCGTCTTCACAAAGAATGCTTGTTG (strain WR)AGACTATCGACAGAGGAACGTCATATATTTTTGGATTACAAGAAA HindIII DTATGGATCCTCTATCCGACTAGAATTAGTCAATCTTATTCAAGCAA fragmentAAACAAAAAACTTTACGATAGACTTTAAGCTAAAATATTTTCTAG DNA,GATCCGGTGCCCAGTCTAAAAGTTCTTTATTACACGCTATTAATCA completeTCCAAAGTCAAGGCCTAATACATCTCTGGAAATAGAATTTACACCTAGAGACAATGAAACAGTTCCATATGATGAACTAATAAAGGAATTGACGACTCTCTCGCGTCATATATTTATGGCTTCTCCAGAGAATGTAATTCTTTCTCCGCCTATTAACGCGCCTATAAAAACCTTTATGTTGCCTAAACAAGATATAGTAGGTTTGGATCTGGAAAATCTATATGCCGTAACTAAGACTGACGGCATTCCTATAACTATCAGAGTTACATCAAACGGGTTGTATTGTTATTTTACACATCTTGGTTATATTATTAGATATCCTGTTAAGAGAATAATAGATTCCGAAGTAGTAGTCTTTGGTGAGGCAGTTAAGGATAAGAACTGGACCGTATATCTCATTAAGCTAATAGAGCCTGTGAATGCAATCAATGATAGACTAGAAGAAAGTAAGTATGTTGAATCTAAACTAGTGGATATTTGTGATCGGATAGTATTCAAGTCAAAGAAATACGAAGGTCCGTTTACTACAACTAGTGAAGTCGTCGATATGTTATCTACATATTTACCAAAGCAACCAGAAGGTGTTATTCTGTTCTATTCAAAGGGACCTAAATCTAACATTGATTTTAAAATTAAAAAGGAAAATACTATAGACCAAACTGCAAATGTAGTATTTAGGTACATGTCCAGTGAACCAATTATCTTTGGAGAGTCGTCTATCTTTGTAGAGTATAAGAAATTTAGCAACGATAAAGGCTTTCCTAAAGAATATGGTTCTGGTAAGATTGTGTTATATAACGGCGTTAATTATCTAAATAATATCTATTGTTTGGAATATATTAATACACATAATGAAGTGGGTATTAAGTCCGTGGTTGTACCTATTAAGTTTATAGCAGAATTCTTAGTTAATGGAGAAATACTTAAACCTAGAATTGATAAAACCATGAAATATATTAACTCAGAAGATTATTATGGAAATCAACATAATATCATAGTCGAACATTTAAGAGATCAAAGCATCAAAATAGGAGATATCTTTAACGAGGATAAACTATCGGATGTGGGACATCAATACGCCAATAATGATAAATTTAGATTAAATCCAGAAGTTAGTTATTTTACGAATAAACGAACTAGAGGACCGTTGGGAATTTTATCAAACTACGTCAAGACTCTTCTTATTTCTATGTATTGTTCCAAAACATTTTTAGACGATTCCAACAAACGAAAGGTATTGGCGATTGATTTTGGAAACGGTGCTGACCTGGAAAAATACTTTTATGGAGAGATTGCGTTATTGGTAGCGACGGATCCGGATGCTGATGCTATAGCTAGAGGAAATGAAAGATACAACAAATTAAACTCTGGAATTAAAACCAAGTACTACAAATTTGACTACATTCAGGAAACTATTCGATCCGATACATTTGTCTCTAGTGTCAGAGAAGTATTCTATTTTGGAAAGTTTAATATCATCGACTGGCAGTTTGCTATCCATTATTCTTTTCATCCGAGACATTATGCTACCGTCATGAATAACTTATCCGAACTAACTGCTTCTGGAGGCAAGGTATTAATCACTACCATGGACGGAGACAAATTATCAAAATTAACAGATAAAAAGACTTTTATAATTCATAAGAATTTACCTAGTAGCGAAAACTATATGTCTGTAGAAAAAATAGCTGATGATAGAATAGTGGTATATAATCCATCAACAATGTCTACTCCAATGACTGAATACATTATCAAAAAGAACGATATAGTCAGAGTGTTTAACGAATACGGATTTGTTCTTGTAGATAACGTTGATTTCGCTACAATTATAGAACGAAGTAAAAAGTTTATTAATGGCGCATCTACAATGGAAGATAGACCATCTACAAGAAACTTTTTCGAACTAAATAGAGGAGCCATTAAATGTGAAGGTTTAGATGTCGAAGACTTACTTAGTTACTATGTTGTTTATGTCTTTTCTAAGCGGTAA VacciniaMDANVVSSSTIATYIDALAKNASELEQRSTAYEINNELELVFIKPPLITL 179 cappingTNVVNISTIQESFIRFTVTNKEGVKIRTKIPLSKVHGLDVKNVQLVDAI enzymeDNIVWEKKSLVTENRLHKECLLRLSTEERHIFLDYKKYGSSIRLELVN catalyticLIQAKTKNFTIDFKLKYFLGSGAQSKSSLLHAINHPKSRPNTSLEIEFTP subunit (D1)RDNETVPYDELIKELTTLSRHIFMASPENVILSPPINAPIKTFMLPKQDI Amino acidVGLDLENLYAVTKTDGIPITIRVTSNGLYCYFTHLGYIIRYPVKRIIDSE sequence (Uni-VVVFGEAVKDKNWTVYLIKLIEPVNAINDRLEESKYVESKLVDICDRI prot IDVFKSKKYEGPFTTTSEVVDMLSTYLPKQPEGVILFYSKGPKSNIDFKIK P04298)KENTIDQTANVVFRYMSSEPIIFGESSIFVEYKKFSNDKGFPKEYGSGKIVLYNGVNYLNNIYCLEYINTHNEVGIKSVVVPIKFIAEFLVNGEILKPRIDKTMKYINSEDYYGNQHNIIVEHLRDQSIKIGDIFNEDKLSDVGHQYANNDKFRLNPEVSYFTNKRTRGPLGILSNYVKTLLISMYCSKTFLDDSNKRKVLAIDFGNGADLEKYFYGEIALLVATDPDADAIARGNERYNKLNSGIKTKYYKFDYIQETIRSDTFVSSVREVFYFGKFNIIDWQFAIHYSFHPRHYATVMNNLSELTASGGKVLITTMDGDKLSKLTDKKTFIIHKNLPSSENYMSVEKIADDRIVVYNPSTMSTPMTEYIIKKNDIVRVFNEYGFVLVDNVDFATIIERSKKFINGASTMEDRPSTRNFFELNRGAIKCEGLDV EDLLSYYVVYVFSKRVaccinia ATGGATGAAATTGTAAAAAATATCCGGGAGGGAACGCATGTCCTT 180 cappingCTTCCATTTTATGAAACATTGCCAGAACTTAATCTGTCTCTAGGTA enzymeAAAGCCCATTACCTAGTCTGGAATACGGAGCTAATTACTTTCTTCA stimulationGATTTCTAGAGTTAATGATCTAAATAGAATGCCGACCGACATGTTA subunit (D12):AAACTTTTTACACATGATATCATGTTACCAGAAAGCGATCTAGATA NucleotideAAGTCTATGAAATTTTAAAGATTAATAGCGTAAAGTATTATGGGA sequenceGGAGTACTAAAGCGGACGCCGTAGTTGCCGACCTCAGCGCACGCA Extracted fromATAAACTGTTCAAACGTGAACGAGATGCTATTAAATCTAATAATC Sequence:ATCTCACTGAAAACAATCTATACATTAGCGATTATAAGATGTTAAC GenBankCTTCGACGTGTTTCGACCATTATTTGATTTTGTAAACGAAAAATAT M15058.1:TGTATTATTAAACTTCCAACTTTATTCGGTAGAGGTGTAATCGATA Vaccinia virusCTATGAGAATATATTGTAGTCTCTTTAAAAATGTTAGACTGCTAAA (strain WR)ATGCGTAAGCGATAGCTGGTTAAAAGATAGCGCCATTATGGTGGC HindIII DTAGTGATGTTTGTAAAAAAAATTTGGATTTATTTATGTCTCATGTT fragmentAAGTCCGTCACTAAGTCTTCTTCTTGGAAGGATGTGAACAGTGTTC DNA,AATTTAGTATTTTAAACAATCCAGTGGATACGGAATTCATTAATAA complete.GTTCTTAGAGTTTTCGAATAGAGTATACGAAGCTCTCTATTACGTTCACTCGTTGCTTTATTCTAGTATGACTTCTGATTCAAAAAGTATCGAAAACAAACATCAGAGAAGACTAGTTAAACTACTGCTGTGA VacciniaMDEIVKNIREGTHVLLPFYETLPELNLSLGKSPLPSLEYGANYFLQISR 181 cappingVNDLNRMPTDMLKLFTHDIMLPESDLDKVYEILKINSVKYYGRSTKA enzymeDAVVADLSARNKLFKRERDAIKSNNHLTENNLYISDYKMLTFDVFRP stimulationLFDFVNEKYCIIKLPTLFGRGVIDTMRIYCSLFKNVRLLKCVSDSWLK subunit (D12):DSAIMVASDVCKKNLDLFMSHVKSVTKSSSWKDVNSVQFSILNNPVD Amino acidTEFINKFLEFSNRVYEALYYVHSLLYSSMTSDSKSIENKHQRRLVKLLL sequence (Uni-prot IDP04318)

Other Embodiments

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

REFERENCES

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We claim:
 1. A chimeric enzyme for synthesizing a capped RNA molecule, said chimeric enzyme comprising at least one capping enzyme and at least one nucleic acid polymerase, wherein said capped RNA molecule comprises at least one chemical modification.
 2. The chimeric enzyme of claim 1, wherein the at least one capping enzyme comprises a Vaccinia capping enzyme comprising a D1 subunit and a D12 subunit or comprises a single subunit capping enzyme.
 3. The chimeric enzyme of claim 2, wherein the D1 subunit is encoded by the nucleic acid sequence SEQ ID NO: 178 and the D12 subunit is encoded by the nucleic acid sequence SEQ ID NO:
 180. 4. The chimeric enzyme of claim 2, wherein the at least one capping enzyme comprises a first region of linked nucleosides encoding the D1 subunit having the amino acid sequence SEQ ID NO: 179 and a second region of linked nucleosides encoding the D12 subunit having the amino acid sequence SEQ ID NO:
 181. 5. The chimeric enzyme of claim 1, wherein the at least one nucleic acid polymerase is selected from the group consisting of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase, a RNA polymerase variant and a DNA polymerase mutant.
 6. The chimeric enzyme of claim 5, wherein the at least one nucleic acid polymerase can utilize at least one chemical modification into the RNA during in vitro transcription.
 7. The chimeric enzyme of claim 5, wherein the at least one nucleic acid polymerase is a T7 RNA polymerase.
 8. The chimeric enzyme of claim 7, wherein the T7 RNA polymerase is encoded by the nucleic acid sequence SEQ ID NO:
 176. 9. The chimeric enzyme of claim 7, wherein the T7 RNA polymerase comprises the amino acid sequence SEQ ID NO:
 177. 10. The chimeric enzyme of claim 5, wherein the at least one nucleic acid polymerase is a RNA polymerase variant and wherein the RNA polymerase variant is a T7 RNA polymerase variant.
 11. The chimeric enzyme of claim 10, wherein the T7 RNA polymerase variant is produced using continuous evolution.
 12. The chimeric enzyme of claim 11, wherein the continuous evolution is phage-assisted continuous evolution (PACE).
 13. The chimeric enzyme of claim 10, wherein the T7 RNA polymerase variant is capable of incorporating at least one chemical modification into RNA during in vitro transcription.
 14. The chimeric enzyme of claim 10, wherein the T7 RNA polymerase variant is characterized as having an increased transcription efficiency through GC-rich regions as compared to a wild type T7 RNA polymerase.
 15. The chimeric enzyme of claim 10, wherein the T7 RNA polymerase variant is characterized as having an increased transcription efficiency to produce messenger RNA as compared to a wild type T7 RNA polymerase.
 16. The chimeric enzyme of claim 5, wherein the at least one nucleic acid polymerase is a DNA polymerase mutant.
 17. The chimeric enzyme of claim 16, wherein the DNA polymerase mutant is a DNA polymerase from Thermococcus gorgonarius (Tgo) comprising at least one mutation.
 18. The chimeric enzyme of claim 17, wherein the DNA polymerase mutant is capable of incorporating at least one chemical modification into RNA during in vitro transcription.
 19. The chimeric enzyme of claim 17, wherein the at least one mutation is located at an amino acid position selected from the group consisting of 93, 141, 143, 403, 409, 485, 657, 658, 659, 663, 664, 669, 671 and 676 of the wild type sequence shown in SEQ ID NO:
 32. 20. The chimeric enzyme of claim 19, wherein the mutation is at amino acid 409 of the wild type sequence.
 21. The chimeric enzyme of claim 20, wherein the mutation is selected from the group consisting of Y409A, Y409G and Y409P.
 22. The chimeric enzyme of claim 21, wherein the mutation is Y409G.
 23. The chimeric enzyme of claim 19, wherein the DNA polymerase from Tgo comprises a mutation at amino acid position 664 of the wild type sequence.
 24. The chimeric enzyme of claim 23, wherein the mutation is selected from the group consisting of E664K, E664L, E664Q and E664R.
 25. The chimeric enzyme of claim 24, wherein the mutation is E664K.
 26. The chimeric enzyme of claim 19, wherein the mutant of the DNA polymerase from Tgo comprises four mutations to the wild type sequence shown in SEQ ID NO: 32; wherein the four mutations are V93Q, D141A, E143A and A485L.
 27. The chimeric enzyme of claim 19, wherein the mutant of the DNA polymerase from Tgo comprises thirteen mutations to the wild type sequence shown in SEQ ID NO: 32; wherein the thirteen mutations are V93Q, D141A, E143A, A485L, P657T, E658Q, K659H, Y663H, E664Q, D669A, K671N, T676I and L403P.
 28. The chimeric enzyme of claim 1, where the at least one capping enzyme comprises a Vaccinia capping enzyme catalytic subunit (D1) and a Vaccinia capping enzyme stimulation subunit (D12), and where the at least one nucleic acid polymerase is T7 RNA polymerase (T7) in one of the following combinations: a) a first polypeptide comprising D1 linked to T7 and a second polypeptide comprising D12; b) a first polypeptide comprising D12 linked to T7 and a second polypeptide comprising D1; c) a single polypeptide comprising D1 linked to D12 linked to T7; d) a single polypeptide comprising D12 linked to D1 linked to T7; e) a single polypeptide comprising T7 linked to D1 linked to D12; or f) a single polypeptide comprising T7 linked to D12 linked to D1, wherein D1, D12 and/or T7 are linked using at least one linker peptide
 29. The chimeric enzyme of claim 28, wherein the at least one linker peptide is selected from the group consisting of (GGGGS)n, LGGGGSGGGGSGGGGSAAA (SEQ ID NO: 173), LSGGGGSGGGGSGGGGSGGGGSAAA (SEQ ID NO: 174), and EGKSSGSGSESKST (SEQ ID NO: 175), wherein n refers to any whole integer.
 30. The chimeric enzyme of claim 1, further comprising an affinity purification tag, optionally a His tag.
 31. The chimeric enzyme of claim 1, wherein the capped RNA molecule is a messenger RNA.
 32. An isolated polynucleotide encoding a chimeric enzyme of any one of claims 1 through
 31. 33. An expression vector comprising the isolated polynucleotide of claim
 32. 34. The expression vector of claim 33 comprising a pEXpress vector or a pET vector.
 35. A host cell comprising the isolated polynucletide of claim 32 or the expression vector of claim 18 or the expression vector of claim
 19. 36. The host cell of claim 35, wherein the host cell is a BL21(DE3) cell.
 37. A method of producing the chimeric enzyme of claim 1, comprising culturing the host cell of claim 35 under conditions sufficient for production of the chimeric enzyme.
 38. The method of claim 37, wherein the host cell is cultured at a temperature below 37 degrees Celsius.
 39. The method of claim 37, wherein solubility of the catalytic subunit of the capping enzyme is increased compared to expression in the absence of the nucleic acid polymerase.
 40. The method of claim 37, further comprising a purification step, optionally selected from the group consisting of an affinity purification, an ion exchange purification, and a size exclusion purification.
 41. A method of synthesizing a capped RNA molecule comprising at least one chemical modification, comprising contacting a DNA template with the chimeric enzyme of claim 1 under conditions sufficient for production of the capped RNA molecule.
 42. The method of claim 41, comprising a single-step reaction for both in vitro transcription and capping.
 43. A kit comprising the chimeric enzyme of claim 1 and instructions for use thereof. 